Method of producing libraries of proteins (options), the method of selection of the desired protein and nucleic acid
The invention relates to biotechnology and can be used for separation of proteins with desired properties from large pools and nucleic acids (TC). The invention provides for hybrids "RNA-protein", which includes the stage of post-translational incubation with a high concentration of monovalent cation. As a monovalent cation use Na+, K+or NH+4at a concentration of from 125 mm to 1.5 M, the Method involves the use and/or bivalent or more high-valent cation, such as Mg+2in a concentration from 25 mm to 200 mm. The method allows to obtain a library of proteins. Anologichny method is used to produce libraries of DNA, which additionally includes a step of generating a DNA molecule from each of the RNA portion of the hybrid. The invention also discloses a method of selection of the desired protein and the TC obtained from libraries and NC proteins, including the production and selection of the desired hybrid RNA-protein with subsequent selection of the desired protein and the TC encoding the desired protein. The use of the invention facilitates the selection of proteins with desired properties, as well as to solve the problem of selection and amplification of the sequence of the protein. 3 N. and 51 C.p. f-n pending application U.S. Szostak and others, reg. No. 09/007005, filed January 14, 1998, claims which arise from the provisional applications U.S., Szostak and others, reg. No. 60/064491, filed November 6, 1997 and is currently revoked, and reg. No. 60/035963, filed January 21, 1997 and now abandoned.
The present invention relates to a method of screening protein.
The present invention was made using funds allocated by the government in accordance with the grant F32 GM17776-01 and F32 GM17776-02. The government has certain rights in this invention.
Current methods of extraction of RNA and DNA molecules based on their functionality. For example, experiments Ellington and Szostak (Nature 346: 818 (1990); and Nature 355: 850 (1992)) and Tuerk & Gold (Science 249: 505 (1990); and J. Mol. Biol. 222: 739 (1991)) have demonstrated that very rarely molecules of nucleic acids with the desired properties (that is, less than 1 out of 1013can be separated from the multicomponent pools of molecules by repeated cycles of selection and amplification. Compared with traditional genetic selection methods, these methods have the advantages that (i) can be skanirovaniya very large pools of candidates (>1015), (ii) the viability of the host and in vivo conditions do not matter, and (iii) the selection can be performed on the at the identification of new RNA and DNA sequences, highly specific functions of binding to protein (see, for example, Tuerk & Gold, Science 249: 505 (1990); Irvine et al., J. Mol. Biol. 222: 739 (1991); Oliphant et al., Mol. Cell. Biol. 9: 2944 (1989); Blackwell et al., Science 250: 1104 (1990); Pollock & Treisman, Nuc. Acids. Res. 18: 6197 (1990); Thiesen & Bach, Nuc. Acids. Res. 18: 3203 (1990); Bartel et al. Cell. 57: 529 (1991); Stormo & Yoshioka, Proc. Natl. Acad. Sci. USA 88: 5699 (1991); and Bock et al., Nature 355: 564 (1992)), the function of binding to small molecules (Ellington & Szostak, Nature 346: 818 (1990); Ellington & Szostak, Nature 355: 850 (1992)) and catalytic functions (Green et al., Nature 347: 406 (1990); Robertson & Joyce, Nature 344: 467 (1990); Beaudry & Joyce, Science 257: 635 (1992); Bartel & Szostak, Science 261: 1411 (1993); Lorsch & Szostak, Nature 371: 31-36 (1994); Cuenoud & Szostak, Nature 375: 611-614 (1995); Chapman & Szostak, Chemistry and Biology 2: 325-333 (1995); and Lohse & Szostak, Nature 381: 442-444 (1996)). A similar scheme selection and amplification of proteins has not been demonstrated.
Brief description of the invention
The aim of the present invention is the implementation of the principles of in vitro selection and in vitro evolution, applied to proteins. The present invention facilitates the selection of proteins with desired properties from large pools fully or partially randomized amino acid sequences. In addition, the present invention allows to solve the problem of selection and amplification of the sequence of the protein by covalent binding encodes political in vitro or in situ transcription/translation, which allows you to generate a protein covalently linked to the 3'end of its own mRNA, i.e., the hybrid RNA-protein". This method is carried out by synthesis and in vitro or in situ translation of the mRNA molecule with a peptide acceptor attached to its 3'-end. One of the preferred peptide acceptors is puromycin, nucleoside analogue, which joins With the end of the growing peptide chain and terminates the transmission. In one of the preferred structures of the DNA sequence is included between the end of the transcript and peptide acceptor, which was designed to cause the stop of the ribosome at the end of the open reading frame, which gives the peptide acceptor (for example, puromycin) extra time to join the growing peptide chain before hydrolysis communication peptidyl-tRNA.
The resulting hybrid RNA-protein allows, if necessary, repeated cycles of selection and amplification, as data on protein sequence can be obtained by reverse transcription and amplification (e.g., by PCR amplification, as well as any other amplification method, including methods of amplification on the basis of RNA, such as 3SR or TSA). Amplifitsirovannykh hybrids "mRNA-protein" for the next cycle of selection. The ability to perform multiple cycles of selection and amplification allows to achieve enrichment of rare molecules and their selection, such as selection of one desired molecule from the pool with 1015members. This, in turn, provides an opportunity to highlight new or improved proteins that specifically recognize virtually any target, or that catalyze the desired chemical reaction.
In accordance with this, in its first aspect the present invention relates to a method of selecting a desired protein, comprising the stage of: (a) producing a population of RNA molecules of candidates, each of which contains a sequence of translation initiation and start-codon, functionally attached to a sequence that encodes a protein candidate, and each of which is functionally attached to the peptide acceptor at the 3'-end of this sequence that encodes a protein candidate; (b) in vitro or in situ translation of sequences encoding a protein candidate, with the production of a population of hybrids candidate RNA-protein; and (C) the selection of the desired hybrid RNA-protein", and thus selection of the desired protein.
In his related aspect, the present invention relates to a method of screening a DNA molecule that encodes a desired protein, etelnost of translation initiation and start-codon, functionally attached to a sequence that encodes a protein candidate, and each of which is functionally attached to the peptide acceptor at the 3'-end sequence that encodes a protein candidate; (b) in vitro or in situ translation of sequences encoding a protein candidate, with the production of a population of hybrids candidate RNA-protein"; (C) selection of the desired hybrid RNA-protein"; and (d) generation of RNA-specific hybrid DNA molecule that encodes a desired protein.
In another related aspect, the present invention relates to a method of selecting a protein with altered function compared to the original protein, where the method involves the following stages: (a) producing a population of RNA molecules candidate populations of DNA-matrices, each DNA-matrix-candidate contains a sequence encoding a protein candidate and different from the sequence that encodes a protein source, where each of these RNA molecules contains a sequence of translation initiation and start-codon, functionally attached to a sequence that encodes a protein candidate, and each of these molecules is functionally attached to the peptide acceptor at the 3'-end; (b) in vitro or in situ broadcast after the boron desired hybrid RNA-protein", having modified the function, and thus selection of a protein with altered function.
In still another related aspect, the present invention relates to a method of screening a DNA molecule that encodes a protein with altered function compared to the original protein, comprising the stage of: (a) producing a population of RNA molecules candidate populations of DNA-matrices candidates, each DNA-matrix-candidate contains a sequence encoding a protein candidate, which is different from the sequence that encodes a protein source, and where each RNA molecule includes a sequence of translation initiation and start-codon, functionally attached to a sequence that encodes a protein candidate, and each RNA molecule is functionally attached to the peptide acceptor at the 3'-end; (b) in vitro or in situ translation of sequences encoding a protein candidate, with the production of a population of hybrids candidate RNA-protein"; (C) selection of hybrid RNA-protein with altered function; and (d) generating from the specified RNA portion of the hybrid DNA molecule that encodes a protein with altered function.
In yet another related aspect, the present invention relates to a method of selecting the desired RNA, vkljuchajuwih of translation initiation and start-codon, functionally attached to a sequence that encodes a protein candidate, and each of which is functionally attached to the peptide acceptor at the 3'-end sequence that encodes a protein candidate; (b) in vitro or in situ translation of sequences encoding a protein candidate, with producerof of population hybrids candidate RNA-protein"; and (C) the selection of the desired hybrid RNA-protein", and thus selection of the desired RNA.
In preferred embodiments of the above methods, the peptide acceptor is puromycin; each of the RNA molecules of candidates also includes a stop sequence, or, in addition, includes a DNA sequence or an analogue DNA sequence covalently linked to 3'-end of the indicated RNA; the population of RNA molecules candidate includes at least 109preferably, at least 1010more preferably, at least 1011, 1012or 1013and most preferably at least 1014different RNA molecules; the specified reaction in vitro-translation carried out in the lysate obtained from eukaryotic cells or parts of it (i.e. this reaction, for example, is carried out in the lysate of reticulocyte or the lysate of wheat germ); ukazanno the parts; the specified stage of the selection process involves the binding of the desired protein to the immobilized binding partner; this stage of the selection process involves performing analysis on the functional activity of the desired protein; the DNA molecule is amplified; this method, in addition, provides for the repetition of the stages of the above selection methods; this method, in addition, provides for transcription of the RNA molecule from a DNA molecule and repeating steps (a)-(d); and after the stage of translation in vitro, this method also involves the step of incubation in the presence of 50-100 mm MD2+; and the hybrid RNA-protein, in addition, includes a sequence of nucleic acids or similar nucleic acid sequence, located directly on the peptide acceptor, which increases flexibility.
In other related aspects, the present invention relates to a hybrid RNA-protein, selected by any of the methods of the present invention; ribonucleic acid, covalently linked by an amide bond with the amino acid sequence in which the amino acid sequence encoded ribonucleic acid; and ribonucleic acid, which includes poderosa protein-candidate, where this RNA is functionally attached to the peptide acceptor (for example, puromycin) at 3'-end of this sequence that encodes a protein candidate.
In its second aspect the present invention relates to a method of selecting a desired protein or desired RNA by enriching the pool of sequences. This method involves performing stages: (a) producing a population of RNA molecules of candidates, each of which includes a sequence of translation initiation and start-codon, functionally attached to a sequence that encodes a protein candidate, and each of which is functionally attached to the peptide acceptor at the 3'-end of this sequence that encodes a protein candidate; (b) in vitro or in situ-broadcast sequence encoding a protein candidate, with the production of a population of hybrids candidate RNA-protein"; (C) contacting the population of hybrids, RNA-protein with a binding partner specific for the RNA portion or protein part of the hybrid RNA-protein" in conditions that primarily facilitate the separation of the complexes of hybrid partner binding-RNA-protein from the unbound members of this population; (d) release related hybrids "RNA-protein"is aniu, specific for the protein parts of the hybrid RNA-protein" in conditions that primarily facilitate the separation of the complex partner binding-RNA-protein from the unbound members of the specified population and, thus, selection of the desired protein and the desired RNA.
In preferred variants of the invention, this method also involves the repetition of steps (a)-(e). In addition, for these repeated stages for selective enrichment of the desired hybrid RNA-protein" can be used the same or other binding partners in any order. In another preferred embodiment, stage (d) involves the use of a binding partner (e.g., monoclonal antibodies) specific for the protein part of the desired hybrid. This stage is preferably carried out after reverse transcription of the RNA portion of the hybrid with the production of DNA that encodes a desired protein. If necessary, this DNA can be isolated and/or PCR-amplified. This method of enrichment can be used for selection of the desired protein, or it can be used to identify a protein with altered function compared to the original protein.
In other preferred embodiments, assise, includes a stop sequence, or, in addition, includes a DNA sequence or an analogue DNA sequence covalently linked to 3'-end of the indicated RNA; this population of RNA molecules candidate includes at least 109preferably, at least 1010more preferably, at least 1011, 1012or 1013and most preferably at least 1014different RNA molecules; the specified reaction in vitro-translation carried out in the lysate obtained from eukaryotic cells or parts of it (this reaction, for example, is carried out in the lysate of reticulocyte or the lysate of wheat germ); the specified reaction in vitro-translation is carried out in an extract derived from prokaryotic cells (e.g. E. coli) or part thereof; specified DNA molecule is amplified; at least one of the binding partners is immobilized on a solid medium; after the stage of translation in vitro, the method additionally includes the stage of incubation, carried out in the presence of 50-100 mm Mg2+; and the hybrid RNA-protein" also includes nucleic acid sequence or similar nucleic acid sequence, u is retina relates to methods of producing libraries (for example, libraries of protein, DNA or RNA hybrids) or to the methods of selecting the desired molecules (e.g. protein, DNA or RNA molecules or molecules that have a specific function, or altered function), including the stage of post-translational incubation in the presence of high salt (including, but not limited to it, the highest salt, which contains a monovalent cation, such as+, NH+4or Na+, divalent cation, such as Mg+2or a combination of both). This incu-repression can be carried out at approximately room temperature or at approximately -20With, and the preferred salt concentration is approximately 125 mm - 1.5 M (more preferably approximately 300 mm - 600 mm) for monovalent cations and approximately 25 mm - 200 mm for divalent cations.
In another related aspect, the present invention relates to kits for carrying out any of the selection methods described in this application.
In his third and last aspect the present invention relates to a microchip, which include an array of immobilized single-stranded nucleic acids, nucleic acids hybridized with hybrids of RNA-protein. Suppose is in the "population" means more than one molecule (for example, more than one RNA, DNA, or a hybrid molecule of RNA-protein"). Because the methods of the present invention facilitate the selection process, which begins, if necessary, with a large number of members of the molecules are candidate components of the "population" of the present invention, preferably consisting of more than 109molecules, more preferably of more than 1011, 1012or 1013molecules, and most preferably of more than 1013molecules.
The term "selection" means, basically, is the separation of one molecule from other molecules in the population. Used herein, the term " stage "selection" means at least 2-fold, preferably 30-fold, more preferably 100-fold, and most preferably 1000-fold enrichment of the population of the desired molecules compared with unnecessary molecules after carrying out this stage of selection. As shown in this application, the phase selection can be repeated any number of times, and in this way can be combined in different types of stages of selection.
The term "protein" means any two or more natural or modified amino acids, linked together by one or more peptide bonds. The terms "protein" and "peptide" are used interchangeably.modifitsirovannyh of ribonucleotides. One example of a modified RNA covered by this term, is phosphorothioate-RNA.
The term "a sequence of translation initiation" means any sequence, with a functional site of accession of the ribosome. In bacterial systems, this field is sometimes called the Shine-dalgarno sequence.
The term "start codon" refers to the three foundations, which are a sign of the start of a sequence that encodes a protein. Usually these grounds are AUG (or ATG), but in this way they can be replaced by any other suitable triplet.
The term "covalently linked" with a peptide acceptor indicates that this peptide acceptor is attached to the "protein coding sequence" or directly through a covalent bond or indirectly through another covalently linked sequence (for example, DNA corresponding to the stop-site).
The term "peptide acceptor" refers to any molecule that can join With the end of the growing peptide chain due to the catalytic activity of the peptidyl transferase function of the ribosome. Typically, such molecules contain (i) the nucleotide or nucleic-like component (e.g., adenosine or similar is nocollate (for example, any of the 20 D - or L-amino acids or any similar (for example, O-methyltyrosine or any of the analogs described by Ellman et al., Meth. Enzymol. 202: 301, 1991) and (iii) the relationship between the two groups (for example, ester, amide or ketone linkage at the 3'-position or less, preferably in the 2'-position); preferably, this connection is, in the main, did not violate the formation of rings of natural ribonucleotidic conformation. Peptide acceptor may also contain a nucleophilic group that can be, but are not limited to, amino group, hydroxyl group or sulfhydryl group. In addition, peptide acceptors may consist of a nucleotide mimetics, amino acid mimetics, or mimetics of the United nucleotide-amino acid structures.
The term "peptide acceptor in the 3'-end of the protein coding sequences" means that the molecule is a peptide acceptor is the final codon of the specified protein coding sequence. This term includes, but is not limited to, a molecule peptide acceptor, which is located directly near the 3'-end of the protein coding sequence, and a molecule that is separated from the target codon is a stop-site). This term also includes structures in which coding or non-coding sequence followed (i.e., with the 3'-end) molecule peptide acceptor. In addition, this term includes, but is not limited to, a molecule peptide acceptor is covalently linked (either directly or indirectly through an embedded sequence of nucleic acids) protein coding sequence, and a molecule that is attached to the protein coding sequence by non-covalent binding, for example by hybridization using the second nucleic acid sequence that binds the 3'-end or near the 3'-end sequence that encodes a protein, which itself is associated with a molecule peptide acceptor.
The term "modified function" refers to any qualitative or quantitative change in the function of the molecule.
The term "stop sequence" means a sequence of nucleic acid, which slows down the level or stops broadcasting under the action of the ribosome.
The term "binding partner" as used in the present description, refers to any molecule that has a specific covalent and who Yu are but not limited to, members of the pairs of antigen/antibody pairs protein/inhibitor pairs of receptor/ligand (e.g., pairs of a cell surface receptor/ligand, such as a pair of hormone receptor/peptide hormone), pairs of enzyme/substrate (e.g., pairs CI-WHI/substrate), pairs lectin/carbohydrate, oligomeric aggregates or heterooligomeric protein pairs DNA-binding protein/binding site with DNA, pairs of RNA/protein and nucleic acid duplexes, heteroduplexes or legirovannykh circuits, as well as any molecule, capable of forming one or more covalent or non-covalent bonds (e.g., disulfide bonds) with any part of the hybrid RNA-protein". The binding partners include, but are not limited to, any of the "reasons for selection, shown in Fig.2.
The term "solid support" means, but is not limited to, any column (or column material), granules, test tube, a Cup for micrometrology, solid particle (for example, agarose or sepharose), microchip (for example, a chip of silicon, silicon glass, or gold) or membrane (for example, the membrane of the liposomes or vesicles), which may contact an affine complex, either directly or indirectly (for example through other intermediate partner is(for example, through receptor or channel).
The term "high salt" means a salt having a concentration of monovalent cation, at least 200 mm, and preferably at least 500 mm or even 1 M, and/or the concentration of divalent cation or a cation of higher valence of at least 25 mm, preferably at least 50 mm, and most preferably at least 100 mm.
The present invention has several significant advantages. First of all, this is the first example of this type of scheme selection and amplification of proteins. The described method allows to solve the problem of the necessity of selection of the nucleotide sequences corresponding to the desired selected proteins (as can only be replicated nucleic acid). In particular, many previously used methods that were allowed to identify proteins that are partially or fully randomized pools envisaged a stage in vivo. Methods of this type are: the technology of monoclonal antibodies (Milstein, Sci. Amer. 243: 66 (1980); and Schultz et al., J. Chem. Engng. News 68:26 (1990)), phage display (Smith, Science 228: 1315 (1985); Parmley & Smith, Gene 73: 305 (1988); and McCafferty et al., Nature 348: 552 (1990)), the use of hybrid peptide-lac-repressor (Cull et al., Proc. Natl. Acad. Sci. USA 89: 18 the methods based on topological relationships between protein and nucleic acid, to see this information about the protein was preserved and could be received in readable form nucleic acids.
In addition, the present invention has advantages compared with the known method broadcast (Tuerk & Gold, Science 249: 505 (1990); Irvine et al., J. Mol. Biol. 222: 739 (1991); Korman et al., Proc. Natl. Acad. Sci. USA 79: 1844-1848 (1982); Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-9026 (1994); Mattheakis et al., Meth. Enzymol. 267: 195 (1996); and Hanes & Pluckthun, Proc. Natl. Acad. Sci. USA 94: 4937 (1997)), a method in which a filter on a specific property of a growing protein chain, which, in addition, forms a complex with the ribosome and mRNA. In contrast to the known method translation method of the present invention is not based on preserving the integrity of the ternary complex of mRNA:ribosome:the growing chain", i.e. complex, which is very fragile, and therefore, the known method is very limited technically possible types of selection.
The method of the present invention also has advantages compared to the method of synthesis of branched proposed by Brenner & Lerner (Proc. Natl. Acad. Sci., USA, 89: 5381-5383 (1992)), which generate hybrids of DNA-peptide, and genetic information theoretically obtained after one cycle of selection. In contrast to the method of branched synthesis method of the present izobreteniya through several cycles of chemical synthesis). In accordance with this method of the present invention allows for repeated cycles of selection using populations of molecules candidate. In addition, in contrast to the extensive method of synthesis, which is mainly limited by the selection is rather short sequences, the method of the present invention can be used for the selection of protein molecules having considerable length.
Another advantage of the present invention is that the method of selection and directed evolution allows the use of very large and complex library sequence candidates. In contrast, existing methods for the selection of proteins, which are based on the stage in vivo, usually limited to a relatively small libraries, with some limited multiplicity. This advantage is particularly important in the selection of functional protein sequences, if we consider that, for example, for a peptide consisting of only 10 amino acids, there is a 1013possible sequences. In classical genetic methods, i.e. methods using hybrid lac-repressor and methods phage display, the maximum multiplicity usually comes is ramineni in the methods of directed evolution, which are that surrounding sequences can be used for a deeper study of this source sequence.
The method of the present invention also differs from previous methods that stage of selection does not depend on the surrounding sequences ("context"). Many other selection schemes specified "context" in which, for example, is expressed protein may significantly affect the nature of the generated library. For example, downregulation of protein will not be able to appropriately expressed in a particular system or may not be properly displayed (for example, on the surface ragovoy particles). An alternative expression of the protein may actually hinder the execution of one or more critical stages in the cycle of selection, for example to affect the viability or infectivity of the phage or the binding of lac-repressor. These negative factors can lead to loss of functional molecules or to restrictions on the nature of the selection procedures that can be used.
Finally, the method of the present invention has advantages as it provides control m antibodies) there is little or no control over the nature of the source pool. In other methods (for example, using lac-hybrid and phage display) pool of candidates must be expressed in the "context" of the hybrid protein. In contrast, hybrid construction "RNA-protein" allow control over the nature of the pools of candidates available for screening. In addition, the size of the pool of candidates may be as large as RNA or DNA pools (~1015members), and is limited only by the extent of the reaction in vitro translation. Creating a pool of candidates depends entirely on the objectives of the experiment; an arbitrary region can be skanirovaniya separately or in the context of" the right of the hybrid protein, and the majority of possible sequences, if not all, can be expressed in pools of candidates hybrids "RNA-protein".
Other characteristics and advantages of the present invention will be apparent from the following detailed description and claims.
Detailed description of the invention
Here is the description of the drawings.
A brief description of the drawings.
In figures 1A-1C schematically illustrates the stages of production of hybrids, RNA-protein. In Fig.1A illustrates the structure of the DNA sample to generate the RNA portion of the hybrid. In Fig.1B support In Fig.2 schematically presents a generalized Protocol selection of the present invention.
In Fig.3 shows schematically the Protocol synthesis minimum translational matrices containing 3'-puromycin. At stage (a) shows the connection of the protective groups to reactive functional groups on puromycin (5'-OH and NH2); as modified groups these groups are suitably protected for use in oligonucleotide synthesis on the basis of phosphoramidite. The specified protected puromycin was attached to the glass with controlled aminohexyl pore size (CPG) via the 2'-OH using standard schemes of joining DNA through its 3'-Oh (Gait, Oligonucleotide Synthesis, A Practical Approach The Practical Approach Series (IRL Press, Oxford, 1984)). In stage (C) the minimum translational matrix (called the "43-R), which contains 43 nucleotide was synthesized using the standard method of chemical synthesis of RNA and DNA (Millipore, Bedford, MA) was protected using the NH4HE and TBAF and subjected to gel purification. This matrix contains 13 of the bases of RNA 5'-end, followed by 29 DNA bases attached to the 3'-puromycin at its 5'-HE. This RNA sequence contains (i) a consensus Shine-dalgarno sequence, complementary to the five bases of the 16S rRNA (Stormo et al., Nucleic Acids bases and (iii) one start codon AUG. This DNA sequence is a dA27dCdCP, where "P" means puromycin.
In Fig.4 schematically shows the preferred way to obtain secure CPG-associated puromycin.
In Fig.5 schematically presents possible methods include methionine in the matrix of the present invention. As shown in reaction (A), this matrix is associated with the ribosome, forming, thereby, a complex of 70S initiation. Fmet-tRNA bound to the P-site and is a couple of reasons associated with the matrix. Puromycin at 3'-end of this matrix enters the a-site by an intramolecular reaction and forms an amide bond with the N-formylmethionine through the peptidyl transferase centre, which thus leads to decelerating tRNA. Extraction of the reaction mixture with phenol/chloroform leads to the formation of a matrix with covalently attached methionine. As shown, the reaction (B) is an undesirable intramolecular reaction of the matrix with oligonucleotides containing puromycin. As mentioned previously, the minimum matrix stimulates the formation of 70S ribosomes containing fmet-tRNA bound to the P site. After this is accomplished through the inclusion of the second matrix in the TRANS-position obtaining covalently tie>-met) in translational matrix. In Fig.6A illustrates the dependence of the reaction of magnesium (MD2+). In Fig.6B shows the basic stability of the product; changes in mobility, shown in this figure, corresponds to the loss of the 5'-RNA sequence 43-R (also known as "Met-matrix") produced DNA-puromycin called 30-R. Retention tags after processing the base corresponds to the formation of the peptide bond between35S-methionine and 3'-puromycin this matrix. In Fig.6C illustrates the inhibition of formation of product in the presence of inhibitors peptidyltransferase. In Fig.6D illustrates the dependence include35S-methionine from the sequence, the coding matrix. In Fig.6E illustrates the dependence of the length of DNA template from including32S-methionine. In Fig.6F illustrates the formation of CIS - or TRANS-product matrices 43-R and 25-R. In Fig.6G illustrates the formation of CIS - or TRANS-product matrices 43-R and 13-R. In Fig.6N illustrates the formation of CIS - or TRANS-product matrices 43-P and 30 P in the system lysate of reticulocyte.
In Fig.7A-7C schematically illustrated designs for nucleotides) (also called "short ICC-matrix') (SEQ ID NO:1). This sequence contains a tag epitope EQKLISEEDL monoclonal antibodies against c-myc (SEQ ID NO:2) (Evan et al., Mol. Cell. Biol. 5: 3610-3616 (1985)), flanked by the 5'-startcode and 3'-linker. This 5'-region contains bacterial Shine-dalgarno sequence identical to the sequence 43-R. This coding sequence was optimized for translation in bacterial systems. In particular, the 5'-UTR 43-P and LP77 contain a Shine-dalgarno sequence, complementary to the five bases of the 16S rRNA (Steitz & Jakes, Proc. Natl. Acad. Sci. USA 72: 4734-4738 (1975)) and located similarly to the sequences of ribosomal protein (Stormo et al., Nucleic Acids Res. 10: 2971-2996 (1982)). In Fig.7B shows LP 154 (legirovannye product length: 154 nucleotide) (also called "long ICC-matrix') (SEQ ID NO:3). This sequence contains the code to generate the peptide used for the selection of antibodies against C-ICC. the 5'-end contains a subset of the above sequence of TMV (indicated by "THOSE"). This 5'-UTR contains 22 nucleotide sequence derived from the 5'-UTR-TMV, covering two direct repeat ACAAAUUAC (Gallie et al., Nucl. Acids. Res. 16: 883 (1988)). In Fig.7C shows pool #1 (SEQ ID NO:4), which is an example of a sequence used for selection as the 3'constant region, required for PCR amplification of this matrix. It is known that this sequence is not part of the epitope binding to the antibody.
In Fig.8 shows a photograph illustrating the synthesis of hybrids of RNA-protein using matrices 43-R, LP77 and LP154 and system broadcast reticulocytes ("reticule.") and wheat germ ("wheat"). In the left part of this figure illustrates the inclusion of35S-methionine in each of these three matrices. In the right part of this figure illustrates the receipt of the products after processing each of these three matrices a And RNase to remove the coding region RNA; shown hybrids "labeled35S-methionine DNA-protein". DNA-part of each of them identical to the oligonucleotide 30-R. Thus, differences in mobility were proportional to the length of the coding regions, which corresponds to the presence of proteins of different lengths in each case.
In Fig.9 shows a photograph illustrating the susceptibility to the protease hybrid RNA-protein synthesized from LP154 and analyzed by electrophoresis in denaturing polyacrylamide gel. Track 1 contains32P-labeled 30-R. Tracks 2-4, 5-7 and 8-10 contain35S-labeled with matrix obtained in financial p is Nesoi a and proteinase K, respectively.
In Fig.10 shows a photograph illustrating the results of the reactions thus using in vitro-translated protein ICC-epitope of 33 amino acids. On track 1 and 2 shows the products of protein translation ICC-epitope and matrices-globin, respectively. On the tracks 3-5 shows the results thus peptide myc-epitope using monoclonal antibodies with the ICC and wash buffer PBS, DB and PBSTDS respectively. On tracks 6-8 shows the same reaction, thus, but use the product broadcast-globin.
In Fig.11 presents a photograph which demonstrated immunoprecipitate of hybrid RNA-protein" from the reaction of translation in vitro. Shown picomole matrix used in this reaction. On tracks 1-4 shows RNK (RNA portion of the hybrid LP154), and on tracks 5-7 shows the hybrid RNA-protein" LP154. After thus using monoclonal antibodies against C-ICC and protein G-sepharose, samples were treated with RNase a and polynucleotide kinase T4, and then loaded on containing urea denaturing polyacrylamide gel to visualize the hybrid. On tracks 1-4 for samples that are either not containing the wasps, corresponding to this hybrid, were clearly visualized. Specified position32P-labeled 30-R, and in the upper part of this figure shows the amount of built-in matrix.
In Fig.12 presents a graph showing the number of hybrid material obtained by the reaction of translation in vitro. The intensity of the bands hybrid, shown on tracks 5-7 Fig.11, and the strip 30-P (selected in parallel on dT25not shown) quantitatively evaluated by phosphor imaging plates and built by the graph of a function of concentration included LP154. The number of selected modified 30-R (left y axis) is directly proportional to the integrated matrix (x-axis) and the number of hybrid linker-peptide (right y axis) was constant. The result of this analysis, it was calculated that a 1 ml sample of the reaction to the broadcast was received ~1012hybrids.
In Fig.13 schematically presents dipropylacetate and dt25the agarose and the ability of these substrates to interact with hybrids RNA-protein" of the present invention.
In Fig.14 presents a picture of demonstrated results consistent selection of hybrids of the present invention. On track 1 porny by RNase. On track 2 LP154 was allocated sequentially using dipropylacetate, and then using dt25-agarose. On track 3 shows the allocation of only using dt25-agarose. The results showed that this product contains a free thiol like penultimate cysteine in the sequence that encodes the ICC-epitope.
In Fig.15A and 15C shown pictures, which demonstrated the formation of hybrid products with matrices-globin analyzed by SDS page with LTOs and trichina (polyacrylamide gel electrophoresis). In Fig.15A shows the inclusion of a35S or no template (lane 1), or matrix Shin--globin (lanes 2-4) or with the matrix LP--globin (tracks 5-7). In Fig.15V (tracks marked as shown in Fig.15A) shows35S-labeled material that has been isolated using affinity chromatography of oligonucleotides. In the absence of 30-P-tail (tracks 2-4) no material was not selected.
In Fig.16A-16C provides diagrams and photographs illustrating the enrichment of the ICC-dzanc towards dzanc pool through in vitro selection. In Fig.16A shows schematically the Protocol that arose for cleaning hybrids matrices from unmodified matrices. Then hybrids "mRNA-peptide was subjected to reverse transcription for inhibition of any secondary or tertiary structure present in the data matrices. Aliquots of each mixture were removed before (Fig.16B) and after (Fig.16C) affinity selection, amplified by PCR in the presence of the labeled primer and hydrolyzed restriction enzyme that splits only ICC-DNA. Included a mixture of matrices were pure ms (lane 1) or the ICC:the pool, 1:20, 1:200 or 1:2000 (tracks 2-4). Because of the preferential translation and reverse transcription ICC-matrix level coming material deviated from the levels of the included material. Enrichment number ICC-matrix in the process of conducting this stage of selection was calculated on the basis of changes in the relationship pool:ms before and after selection.
In Fig.17 shows a photograph illustrating the translation ICC-RNA-matrices. We used the following linkers: tracks 1-4, dA27dCdCP; lanes 5-8, dA27rCrCP; and lanes 9-12, dA21C9C9C9dAdCdCP. On each track the concentration of the RNA matrix was 600 nm, a35S-Met used for tagging. The reaction conditions were as follows: lanes 1, 5 and 9 - 30C, 1 h; lanes 2, 6 and 10 - 30With, 1 hour, -20With 16 hours with 50 mm Mg2+. In this figure "A" represents free peptide, and "In" represents the hybrid mRNA-peptide".
In Fig.18 shows a photograph illustrating the translation ICC-RNA-matrices, labeled32S. Used by the linker was da21With9With9With9ddd. The broadcast was carried out for 90 minutes at 30C, and incubation was carried out at -20With in 2 days without additional quantity of Mg2+. The concentration of mRNA-matrices was 400 nm (lane 3), 200 nm (lane 4), 100 nm (lane 5) and 100 nm (lane 6). On track 1 shows the hybrid mRNA-peptide" labeled35S-Met. On track 2 shows mRNA marked32P. On the track 6 the reaction was carried out in the presence of 0.5 mm cap analogue.
In Fig.19 shows a photograph illustrating the translation ICC-RNA-matrix using a lysate obtained from Ambion (lane 1), Novagen (lane 2) and Amersham (lane 3). Used by the linker was dA27dCdCP. The concentration matrix was 600 nm, a35S-Met used for tagging. The broadcast was carried out at 30C for 1 h, and incubation was carried out at -20In Fig.21 shows a graph illustrating the analysis of competitive binding with synthetic ICC-peptides.
In Fig.22 schematically illustrates the amino acid sequence of the 12 selected peptides from random 27-dimensional library.
In Fig.23 shows a photograph illustrating the effect of the length of the linker on the formation of the hybrid. In this figure, the ICC is a matrix containing long linkers [N]=13, 19, 25, 30, 35, 40, 45 or 50 nucleotides (dA10-47dCdCP), were analyzed for the formation of hybrids by electrophoresis in SDS page with LTOs. Also shown is a flexible linker F (dA21[C9]3dAdCdCP). The translation was performed using a 600 nm matrix with 30C for 90 minutes and then was added 50 mm MD2+and incubated at -20With in two days.
In Fig.24 shows a photograph illustrating a joint broadcast of the ICC and mRNA-Fpsu. This figure of 200 nm RNAFazy (RNC) and/or 50 nm myc-PHK (PHK152) containing rigid linker F (dA21[C9]3dAdCdCP), broadcasted using the [35S]-Met. Then dobavlialsea "ICC-matrices with proteinPpaz) bands were observed.
What follows is a description of the General method of selection of proteins with desired functions, with the use of hybrids, in which these proteins are covalently linked to their own informational RNA. These hybrids "RNA-protein" are synthesized by in vitro - or in vivo-broadcast pools of mRNAs containing peptide acceptor attached to their 3'-ends (Fig.1B). In one preferred embodiment of the present invention after penetrating reciting open reading frame of the transcript of the ribosome when it reaches a certain stop-site, stops, and acceptor group occupies the ribosomal A-site and agrees to the growing peptide chain from the peptidyl-tRNA in P-site, resulting in the formation of a hybrid RNA-protein" (Fig.1C). Covalent bond between the protein and RNA (in the form of an amide bond between the 3'-end of mRNA and C-end of the protein that it encodes) allows after selection by reverse transcription of this RNA to obtain genetic information in a given protein, and to amplify this information (e.g. by PCR). After receiving hybrid selection or enrichment is performed on the basis of the properties of the hybrid mRNA-protein, or, alternatively, it may be the conduct is such or influence odnotsepochechnoi RNA on this selection. When using mRNA-protein selected hybrids can be tested in order to determine which component (protein, RNA or both molecules) provide the desired function.
In one preferred variation puromycin (such tyrosinekinase) acts as an acceptor for joining the growing chain of the peptide to its mRNA. Puromycin is an antibiotic that works by termination of elongation of the peptide chain. As mimetica aminoacyl-tRNA he acts as a universal inhibitor of protein synthesis by binding to A-site acceptance of the growing peptide chain and the separation of ribosomes (Kd=10-4M) (Traut & Monro, J. Mol. Biol. 10: 63 (1964); Smith et al., J. Mol. Biol. 13: 617 (1965)). One of the most attractive features of puromycin is that it forms a stable amide bond with the growing peptide chain, allowing, thus, to obtain more stable hybrids than the potential acceptors that form unstable ester linkages. In particular, the molecule peptidyl-puromycin contains a stable amide bond between the peptide and O-methyltyrosine part of puromycin. O-methyltyrosine, in turn, is connected through a stable amide bond with the 3'-aminogroups tRNA-like structure at the 3'-end of mRNA, as well as other compounds that are similar to puromycin. Such compounds include, but are not limited to, any compound which has an amino acid bond with adenine or adenine-like compound, such as amino acid-nucleotide, i.e. phenylalanyl-adenosine (A-Phe), tyrosyl-adenosine (And Tight) and alanine-adenosine (A-l), as well as patterns with amide bond, such as i.e. phenylalanyl-3'-deoxy-3'-aminoadenosine, alanyl-3'-deoxy-3'-aminoadenosine and tyrosyl-3'-deoxy-3'-aminoadenosine; and in any of these compounds can be used any natural L-amino acids or their analogues. In addition, in the present invention can be also used complex tRNA-like conjugate 3'-structure-puromycin.
The preferred sampling scheme of the present invention shown in Fig.2. The stages included in this sampling scheme, usually as follows.
Stage 1. Obtaining DNA template
Because this stage is aimed at generating hybrids of RNA-protein" of the present invention, the synthesized RNA portion of the hybrid. This can be done by direct chemical synthesis of RNA or, more often, by transcription of the corresponding double-stranded DNA template.
Such DNA's DNA methods of chemical synthesis or in both). In principle, for this purpose can be used any method that allows to produce one or more matrices containing a known, random, randomised or mutagenically sequence. In one of the concrete ways synthesize oligonucleotide (e.g., containing an arbitrary base) and before its transcription is subjected to amplification (e.g., by PCR). Chemical synthesis can also be used for the production of cluster random sequences, which can then be embedded in the middle of a sequence that encodes a known protein (see, for example, Chapter 8.2 Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons and Greene Publisching Company, 1994). Specified the latter method gives a high density of mutations at the desired specific site in the protein.
The alternative to a complete randomization of the sequence of the DNA template is incomplete randomization, and the pool is synthesized in this way is generally referred to as "doped" with a pool. An example of such a method, performed on RNA-sequencing is the method described, for example, Ekiand et al. (Nucl. Acids Research 23: 3231 (1995)). Incomplete randomization can be assests which contained an excess of one of the base and a small amount of each of the other grounds; and the implementation of this method can be achieved by careful control of the concentration of bases and the frequency of the desired mutations. Partially randomized pools can also be generated by the method of conducting error-prone PCR, for example, described Beaudry & Joyce (Science 257: 635 (1992)) and Bartl & Szostak (Science 261: 1411 (1993)).
To create DNA constructs also there are many methods, which proceed from the known sequence, and then create mutagenically DNA pool. Examples of such techniques are described in Ausubel et al. (see Chapter 8); Sambrook et al. (Molecular Cloning: A Laboratory Manual, chapter 15, Cold Spring Harbor Press, New York, 2nded. (1989); Cadwell et al. (PCR Methods and Applications 2: 28 (1992)); Tsang et al. (Meth. Enzymol. 267: 410 (1996)); Reidhaar-Olsen et al. (Meth. Enzymol. 208: 564 (1991)); and Ekiand & Bartel (Nucl. Acids. Res. 23: 3231 (1995)). Random sequences can be generated by the method of "shuffling" as described by Stemmer (Nature 370: 389 (1994)). And finally, a series of two or more heterologous genes may be subjected to recombination in vitro to obtain the original library (Crameri et al. Nature 391: 288-291 (1998)).
ORS can be constructed from arbitrary sequences in a number of ways, depending on the selected codons. Stop codons in the open reading frame preferably be avoided. Can be apnoe the number of stop codons (3/64=4.7% codon), which may be unacceptably high for all but the shortest libraries. Such libraries also contain rarely used codons, which can sometimes lead to insufficient broadcast. NNG codons/S give a somewhat lower frequency of stop codons (1/32+3.1% in codon), but provide access to the "best" codons for all 20 amino acids in the broadcast system of mammals. Codons NNG/C are less than optimal for their use in bacterial systems broadcast, where is the best codons ending in a or T in 7 cases (AEGKRTV). There are several solutions that give a very low frequency of stop codons (~1,0%), where the amino acid content similar to that observed in globular proteins, using three different nucleotide mixtures, codons N1N2N3(LaBean & Kauffman, Protein Science 2: 1249-1254 (1993)) (and cited here). And finally, for libraries configurations in accordance with the type of amino acids that can be used almost infinite number polyrational strategies design. So, for example, hydrophobic (h) or polar (p) amino acids can be selected using codons NTN or NAN, respectively (Beasley & Hecht, J. Biol. Chem. 272: 2031-2034 (1997)). These structures can be in the gif">-fold (phphph...).
ORS constructed from synthetic sequences may also contain stop codons, derived from insertions or deletions in synthetic DNA. These defects can have negative consequences caused by alterations in the translational reading frame. Evaluation of a number of pools synthetic gene constructed from synthetic oligonucleotides showed that insertions and deletions occur with a frequency of ~0.6% at the position or 1.8% at codon. The exact frequency of these events varies, and it is assumed that it depends on the source and the length of the synthetic DNA. In particular, the longer the sequence, the higher the frequency of insertions and deletions (Haas et al., Current Biology 6: 315-324 (1996)). To reduce shifts the reading frame in the LFS can be used a simple solution that is designed to work with relatively short segments of synthetic DNA (80 nucleotides or less), which can be purified to homogeneity. Longer ORS can then be generated by restriction and ligation of some of the more short sequences.
For optimizing the selection of the present invention can also be modified sequence and structure at the 5' and 3'ends which sees the insertion of randomized domains in a matrix, located near the corresponding end, with subsequent selection. These screening stage can serve (i) to maximize the number of obtained hybrid (and thus, to maximize the plurality of libraries) or (ii) to obtain the optimized sequences broadcast. In addition, the method can be mainly applied in combination with mutagenic PCR to optimize translational matrices as in the coding and non-coding regions.
Stage 2. Generating RNA
As mentioned above, the RNA portion of the hybrid RNA-protein can be synthesized by the method of chemical synthesis using standard methods of oligonucleotide synthesis. Alternatively, and particularly if you use a longer RNA sequences, RNA portion generated by in vitro transcription DNA-matrix. In one preferred method for the enzymatic generation of RNA chains using T7 polymerase. Transcription is usually carried out in the same volume as the PCR reaction (PCR DNA obtained from 100 μl of reaction is used for 100 ál-transcription). If necessary, this RNA can be generated from the 5'-cap using a large molar excess of m7GpppG with respect to GT the Xia, but not limited to, SP6, T3 RNA polymerase of E. coli (described, for example, Ausubel et al. (see Chapter 3). In addition, the synthesized RNA can be entirely or partially modified RNA. In one of the concrete examples poportional-RNA can be produced (for example, by T7 transcription) using modified ribonucleotides and standard methods. Such modified RNA have the advantage that they are stable against the action of nucleases. Then the samples are full-size purify RNA from the transcription reaction mixture, as described earlier, using SDS page containing urea, followed by desalting on a NAP-25 (Pharmacia) (Roberts and Szostak, Proc. Natl. Acad. Sci., USA, 94: 1227-12302 (1997)).
Stage 3. Ligation puromycin matrix
Then puromycin (or any other suitable peptide acceptor) covalently associated with the sequence matrix. This stage can be carried out using DNA T4 ligase to attach puromycin directly to RNA sequence, or preferably puromycin can be joined by DNA"shunt" using DNA ligase T4 or any other enzyme capable of linking together two of the nucleus is inane puromycin-like compounds to the RNA can also be used tRNA synthetase. So, for example, i.e. phenylalanyl-tRNA synthetase binds phenylalanine with, i.e. phenylalanyl-tRNA molecules containing 3'-amino group, generating RNA molecules with puromycin-like 3'-ends (Fraser & Rich, Proc. Natl. Acad. Sci., USA, 70: 2671 (1973)). Other suitable peptide acceptors that can be used are, but are not limited to, any of the compounds having the amino acid associated with adenine or adenine-like compound, such as amino acid-nucleotide, i.e. phenylalanyl-adenosine (A-Phe), tyrosyl-adenosine (And Tight) and alanyl-adenosine (A-Ala), as well as related amidon patterns, such as i.e. phenylalanyl-3'-deoxy-3'-aminoadenosine, alanyl-3'-deoxy-3'-aminoadenosine and tyrosyl-3'-deoxy-3'-aminoadenosine; and in any of these compounds can be used any natural L-amino acids or their analogues. A number of peptide acceptors are described, for example, Krayevsky & Kukhanova, Progress in Nucleic Acids Research and Molecular Biology 23: 1 (1979).
Stage 4. The generation and selection of hybrids, RNA-protein
To generate hybrids "RNA-protein" can be used in any in vitro or in situ system broadcast. As shown below, are preferred eukaryotic systems, and particularly preferred are two systems, namely the system of embryos PSH is, each system broadcast, which allows you to get a hybrid RNA-protein and which does not have a significant destructive effect on the RNA portion of the hybrid. In addition, to reduce the degree of destruction of RNA in any of these systems in translational reaction mixture can be enabled antisense oligonucleotides that block the degradation; and such oligonucleotides specifically hybridize with sequences and cover sequence in the RNA part of the molecule, which stimulates the destruction (see, for example, Hanes & Pluckthun, Proc. Natl. Acad. Sci., USA, 94: 4937 (1997)).
As mentioned above, the present invention can be used any number of eukaryotic systems broadcast. Such systems include, but are not limited to, lysates of yeast, ascites, tumor cells (Leibowitz et al., Meth. Enzymol. 194: 536 (1991)) and Xenopus oocytes. Suitable bacterial systems in vitro translation are, but not limited to the system described by Zubay (Ann. Rev. Genet. 7: 267 (1973)); Chen & Zubay (Meth. Enzymol. 101: 44 (1983)); and Ellman (Meth. Enzymol. 202: 301 (1991)).
In addition, reactions broadcast can be performed in situ. In one specific example, the transmission may be carried out by injecting mRNA into Xenopus egg standard methods.
After generating hybrids of RNA-protein onebyone used methods of protein purification. As shown below, for example, purification of the hybrid can be facilitated by using appropriate chromatographic reagents, such as dt25-agarose or thiopropyl-sepharose. However, cleaning can also, or alternatively, to provide clearance on the basis of the RNA portion of the hybrid; the methods of treatment described, for example, Ausubel and others (see above, Chapter 4).
Stage 5: selection of the desired hybrid RNA-protein
The selection of the right hybrid RNA-protein can be carried out in any manner suitable for the selective separation or highlight the desired hybrid of population hybrids candidates. Examples of separation methods include, but are not limited to, selective binding, for example, with a binding partner, which is directly or indirectly immobilized on a column, sphere membrane or other solid media; and immunoprecipitated using antibodies specific for the protein part of this hybrid. The first of these methods enables the use of immobilized motive for the selection, which may consist of molecules of any type, with which it is associated. The list of possible molecules for the selection of motifs is shown in Fig.2. The selection can also be implemented on the basis of ispolzavolai candidate, or based on another type of interaction with the hybrid molecule. In addition, proteins can be selected on the basis of their catalytic activity in a manner analogous to the described Bartel and Szostak for separation of RNA enzymes (see above); and in accordance with the specific method of the desired molecule is chosen based on their ability to bind with the molecules of the target, after which these functional molecules emit, based on the presence of the specified target. The present invention provides for the use of selection schemes for allocation of new or improved catalytic proteins using the same method or any other method of functional selection.
In addition, as described in this application, the selection of the desired hybrid RNA-protein" (or DNA copies) may be facilitated by enrichment with this hybrid pool of molecules candidate. To implement this optional enrichment of the population of hybrids of candidate RNA-protein" is subjected to contact with the binding partner (for example, with one of the above partners linking), which is specific for the RNA part or for the protein part of this hybrid, which basically allows the separation of complex partner predpochtitelno includes, at least two successive stages of enrichment, one of them undertaking the selection of hybrids with the use of a binding partner specific for the RNA portion and the other carried out the selection of hybrids with the use of a binding partner that is specific for this protein. In addition, if the stage of enrichment, directed to the same portion of the hybrid (for example, the protein part), I repeat, it is preferable to use other binding partners. In one specific example described in this application, the population of molecules enrich the desired hybrids first, using the binding partner specific for the RNA portion of the hybrid, and then, in two successive stages, with two other partners on binding, each of which is specific for the protein part of the hybrid. And again, these complexes can be separated from the components of the sample by any standard methods of separation, including, but not limited to, affinity column chromatography, centrifugation or immunoprecipitation.
Moreover, the elution of the hybrid RNA-protein complex enrichment (or selection) can be carried out by a number of methods. For example, the or nonspecific chemical elution. For this stage facilitates the separation of the components from each other or from the associated solid media in a relatively nonspecific manner by destroying non-covalent bonds between these components and/or between the components and solid media. As described in this application, one typical example of a denaturing reagent or non-specific reagent for chemical elution is 4% HOAc/H2O. Other examples of denaturing reagents or reagents for non-specific chemical elution are guanidine, urea, highly concentrated salt, detergent or any other means by which can be mostly removed non-covalent adducts. An alternative method can be used specific chemical elution, which uses a chemical substance that promotes the specific release of hybrid molecules. In one specific example, if the linker "leg" of the desired hybrid protein contains one or more disulfide bonds, the associated hybrid aptamers can be polyuretane by adding, for example, DTT, which leads to the restoration of the disulfide bond and release tie complexes; such methods can selectively release the components of the complex by adding an excess of one member of this complex. For example, in the selection by ATP-binding elution is carried out by adding an excess of ATP to the mixture for incubation. And finally, can be carried out stage enzymatic elution. In accordance with this method, the related molecule or Exo-gene attached protease (or other suitable hydrolytic enzyme) breaks down and releases either the target or enzyme. In one particular example, the website of the accession of the protease can be included in any of the components of the complex, and related molecules elute by adding protease. Alternative when the catalytic selection elution can be performed as a stage of the selection process for the selection of molecules capable of self-separation (e.g., by cleavage from the solid media.
Stage 6. Generating a DNA copy of the RNA sequence using reverse transcriptase
If necessary, a DNA copy of the selected sequence of RNA hybrid can be easily obtained by reverse transcription of this RNA sequences using standard methods of selection or enrichment (e.g., as shown in Fig.16) or after this stage. An alternative process of reverse transcription can be carried out before allocating hybrid of translational in vitro or in situ mixture.
Then DNA template amplified either in the form of partial or full-length double-stranded sequence. Preferably at this stage, the full-length DNA-matrix generated using the appropriate oligonucleotides and PCR amplification.
These stages, as well as the reagents and methods of implementation of these stages is described in detail in the following specific examples. These examples are provided to illustrate the present invention and should not be construed as limiting thereof.
Generating matrices for hybrids "RNA-protein"
As shown in Fig.1A and 2, the sampling scheme of the present invention preferably allows the use of double-stranded DNA template, which includes a number of desired items. The first of these elements is the promoter used in combination with the appropriate RNA polymerase for the synthesis of mRNA. As shown in Fig.1A, in the present description, it is preferable for the T7 promoter, although it may be used any promoter capable of regulating the synthesis of linear double-stranded GNCRC, above the site of translation initiation. In Fig.1A shows the preferred 5'UTR (called "THOSE"), which is a deletion mutant 5'-untranslated region of tobacco mosaic virus, and in particular corresponds to the grounds, located directly to the 5'-end from the site of translation initiation TMV; where the sequence of this UTR is a rGrGrG
rArCrA rArUrU rArCrU rArUrU rUrArC rArArU rUrArC rA (where the first 3 nucleotide G is built to enhance transcription) (SEQ ID NO:5). Can be used any other suitable 5'UTR (see, for example, Kozak, Environ. Rev. 47: 1 (1983); Jobling et al., Nature 325: 622 (1987)).
The third element, shown in Fig.1A, is the site of translation initiation. Basically, it is a codon AUG. However, there are examples where natural coding sequences instead of AUG there are other codons, and these codons can be used in the sampling scheme of the present invention. On the efficiency of translation is affected by a specific sequence context surrounding this codon (Kozak, Microbiological Reviews 47: 1-45 (1983); and Kozak, J. Biol. Chem. 266: 19867-19870 (1991)). Sequence 5'RNNAUGR gives a good context of the start codon for most sequences, is preferred as the first purine (-3) is a, And
The fourth element of Fig.1A is an open reading frame protein (called OCR), which encodes this protein. This open reading frame may encode any natural, arbitrary, randomised, mathenesserlaan or completely synthetic sequence of the protein. The most salient feature of the LFS and the adjacent 3'-constant region is that none of them contains stop codons. The presence of stop codons should lead to premature termination of protein synthesis, which prevents the formation of a hybrid.
The fifth element shown in Fig.1A is a constant 3'-region. This sequence facilitates PCR amplification of sequences of pools and ligation parametersetname of the oligonucleotide to the mRNA. If necessary, this area may also include a stop site sequence which causes the stop of the ribosome, which gives the acceptor part (for example, puromycin additional time period for acceptance of the growing peptide chain from the peptidyl-tRNA; this stop-site are discussed further below.
For the development of the methodology of the present invention hybrids "RNA-protein" were first generated with use of the am. First, the matrix of this size can be easily obtained by chemical synthesis. And, secondly, a small open reading frame makes it easy to analyze important features of this reaction, including the effectiveness of binding heterogeneity of all, the dependency matrix and the accuracy of the translation.
The basic design used to generate the test hybrids "RNA-protein". The molecule consists of an mRNA containing the Shine-dalgarno sequence (SD) for initiation of translation, which contains deletions in the 3 bases in the SD sequence from ribosomal protein L1 and which is complementary to the 5 bases of the 16S rRNA (i.e rGrGrA rGrGrA rGrGrA rA) (SEQ ID NO:6) (Stormo et al., Nucleic Acids Research 10: 2971-2996 (1982); Shine & Dalgarno, Proc. Natl. Acad. Sci. USA 71: 1342-1346 (1974); and Steitz & Jakes, Proc. Natl. Acad. Sci. USA 72: 4734-4738 (1975)), (ii) the start codon AUG, (iii) DNA-linker acting as a stop-site (i.e., 5'-(dA)27), (iv) dCdC-3' and (v) a 3'-puromycin (P). Poly-dA sequence was chosen because of the known low content matrix tRNA in the a site (Morgan et al., J. Mol. Biol. 26: 477-497 (1967); Ricker & Kaji, Nucleic Acids Research 19: 6573-6578 (1991)) and is constructed so that it functioned as a good stop-site. The length of the oligo dT-linker is chosen so as to span the distance between fruits for in order to facilitate the binding of puromycin a-site of the ribosome.
Chemical synthesis of minimal matrix 43-P
For the synthesis of structures 43-R (shown in Fig.3) puromycin first attached to the solid carrier in such a way that they are compatible with standard phosphoramidite method of chemical synthesis of oligonucleotides. The scheme of synthesis for a given oligonucleotide is schematically shown in Fig.3 and described in more detail below. To attach puromycin to solid media from glass with adjustable pore size (CPG), the amino group protecting trifluoracetyl group described in Applied Biosystems User Bulletin #49, for a DNA synthesizer model 380 (1988). Then protect 5 ON using standard DMT-C1-method (Gait, Oligonucleotide Syntesis a practical approach The Practical Approach Series (IRL Press, OxFord, 1984), and joining aminohexyl-CPG through 2 ON carry out exactly the same method as the method which should be used 3 ON to join deoxynucleoside (see Fig.3 and Gait, see above, p. 47). After this 5' DMT-CPG-bound protected puromycin can be used for chain elongation phosphoramidite monomers. The synthesis of the oligonucleotide in the direction 3'5' is carried out in the following order: (i) 3'-Alnost design 43-R shown below.
Synthesis of CPG-puromycin
Synthesis of protected CPG-puromycin is carried out in accordance with the General method used to deoxynucleosides described previously (Gait, Oligonucleotide Synthesis a practical approach The Practical Approach Series (IRL Press, OxFord, 1984)). The main starting points are the choice of an appropriate N-blocking group, joining puromycin 2 ON to a solid carrier and carrying out the reactions of addition to solid media. In the latter case, the above reaction is carried out at very low concentrations of activated nucleotide, because this material is significantly more valuable than solid media. The resulting output (~20 µmol/g of carrier) is sufficient given the conditions of the reaction breeding.
Synthesis of N-triftoratsetofenona
267 mg (0,490 mmol) puromycinHCl was first converted into the free base by dissolving in water with the addition of carbonate buffer, pH 11, and extraction (3) in chloroform. The organic phase is evaporated to dryness and weighed (242 mg, 0,513 mmol). Then the free base was dissolved in 11 ml of dry pyridine and 11 ml of dry acetonitrile was added with stirring 139 μl (2.0 mmol) of triethylamine (TEA; Fluka) and 139 μl (1.0 mmol) of the anhydride triperoxonane has not been spent, as it was shown by thin layer chromatography (TLC) (93:7, chloroform/Meon) (a total of 280 μl). This reaction proceeded for one hour. At this stage, using thin-layer chromatography revealed two bands, both of which had greater mobility than the original material. Processing of the reaction mixture NH4OH and water led to a decrease in product to one lane. After chromatography on silica gel (93:7, chloroform/Meon) received 293 mg (0,515 mmol) of the product, N-TFA-Pur. The product of this reaction is shown schematically in Fig.4.
Synthesis of N-TRIFLUOROACETYL-5'-DMT-puromycin
The product of the above reaction was divided into aliquots and evaporated 2 together with dry pyridine to remove the water. For testing a variety of reaction conditions to prepare a lot of tubes. For reactions on a small scale 27.4 mg (48.2 mmol) of N-TFA-Pur was dissolved in 480 μl of pyridine containing 0.05 EQ. DMAP and 1.4 EQ. TEA. To this mixture was added 20.6 mg of dimethoxyethane (60 μmol) and the reaction mixture was left under stirring until complete reaction. The reaction was stopped by adding to the solution an equal volume of water (approximately 500 ml). Since this reaction was successful, it was carried out a large-scale reaction. In particular, 262 mgrid (Sigma). After approximately two hours, was added 50 mg (0.3 EQ.) dimethoxytritylCL (DMTCl), and left to undergo the reaction for another 20 minutes. The reaction was stopped by adding 3 ml of water, and the reaction mixture was subjected to co-evaporation (3) CH3The JV. Then the reaction mixture was purified with a mixture of chloroform/Meon, 95:5, on a column of diameter 2 mm with 100 ml of silicon dioxide (dry). Due to incomplete cleaning of the second identical column was suirable a mixture of chloroform/Meon, 97:5:2,5. The total yield amounted to 325 mg or 0,373 mmol (or yield 72%). The product of this reaction is shown schematically in Fig.4.
Synthesis of N-TRIFLUOROACETYL, 5'-DMT, 2'-succinyl-puromycin
In the reaction on a small scale 32 mg (37 μmol) of product, synthesized as described above was combined with 1.2 EQ. DMAP dissolved in 350 μl of pyridine. To the resulting solution was added 1.2 equivalents of succinic anhydride in 44 μl of dry CH3CN and leave at night for mixing. Thin layer chromatography revealed a small amount of remaining starting material. In large-scale reactions 292 mg (336 mmol) previously obtained product was combined with 1.2 EQ. DMAP in 3 ml of pyridine. To the resulting solution was added 403 μl of 1 M lantern which I again showed a small amount of remaining starting material. These two reaction mixtures were combined and added to 0.2 EQ. DMAP and succinate. The resulting product is evaporated together with toluene (1x) and dried in high vacuum to obtain a yellow foam. Then added CH2CL2(20 ml) and the resulting solution was twice extracted with 15 ml of 10% ice citric acid and then was extracted twice with purified water. The obtained product was dried, and was again dissolved in 2 ml of CH2Cl2and precipitated with stirring by adding 50 ml of hexane. Then the product was subjected to vortex-mixed and centrifuged at 600 rpm for 10 minutes in a laboratory centrifuge. A large part of the elution solvent was removed and the remaining product was dried, first in a low vacuum and then under high vacuum in a drying Cabinet. The output of this reaction product was approximately 260 µmol for sequential output of ~70%.
Synthesis of N-TRIFLUOROACETYL-5'-DMT, 2'-succinyl-G-puromycin
The product obtained in the preceding stage, dissolved in 1 ml of dioxane (Fluka), and then in a mixture of 0.2 ml of dioxane/0.2 ml of pyridine. To the resulting solution was added 40 mg p-NITROPHENOL (Fluka) and 140 mg of dicyclohexylcarbodiimide (DCC; Sigma), and the reaction mixture was left for 2 hours to undergo reaction. Nerastvorim cistella with adjustable aminohexyl pore size (CPG), suspended in 22 ml of dry DMF, and stirred overnight. Then the resin is washed with DMF, methanol and ether and dried. The resulting resin containing at 22.6 µmol of trityl on g, were thoroughly analyzed within the acceptable range for the given media type. Then the media was kepirohi by incubation with 15 ml of pyridine, 1 ml of acetic anhydride and 60 mg of DMAP in 30 minutes. The resulting material on the column gave a negative result (absence of color) ninhydrin test in contrast to the results obtained before blocking, in which this material was produced dark blue color. The product of this reaction is shown schematically in Fig.4. Alternative puromycin-CPG can be obtained industrially.
Synthesis of conjugate mRNA-puromycin
As discussed above, puromycin associated with the oligonucleotide, can be used in either of two ways to generate the conjugate mRNA-puromycin, which acts as a translational matrix. For very short open reading frames of puromycin-oligonucleotide typically lengthen chemical method using RNA or DNA monomers, resulting in a get a fully synthesized matrix. If you require a longer odrt and DNA ligase T4, as described Moore & Sharp (Science 256: 992 (1992)).
In vitro translation and testing hybrids "RNA-protein"
Matrix generated as described above were translated in vitro using both bacterial and eukaryotic systems in vitro-broadcast as follows.
In vitro translation of minimal matrices
43-P and related conjugates RNA puromycin was added in several different systems in vitro-broadcast, including: (i) the S30 system, derived from E. coli MRE600 (Zubay, Ann. Rev. Genet. 7: 267 (1973); Collins, Gene 6: 29 (1979); Chen & Zubay, Methods Enzymol, 101: 44 (1983); Pratt, in reduced and Translation: A Practical Approach, B. D. Harmmes, S. J. Higgins, Eds. (IRL Press, Oxford, 1984), pp. 179-209; and Ellman et al., Methods Enzymol. 202: 301 (1991)), obtained as described by Ellman et al. (Methods Enzymol. 202: 301 (1991)); (ii) ribosomal fraction, derived from the same strain and obtained as described Kudlicki et al. (Anal. Chem. 206: 389 (1992)); and (iii) the S30 system, derived from BL21 E. coli and obtained as described Lesley et al. (J. Biol. Chem. 266: 2632 (1991)). In each case we used the premix obtained as described Lesley et al. (J. Biol. Chem. 266: 2632 (1991)), and incubation was carried out for 30 minutes.
The study of the nature of this hybrid
Matrix 43-R was first tested with extracts broadcast S30 from E. coli. In Fig.5 (Reaction A) illustrates the desired intramolecular (CIS) reaction, where trovano the inclusion of35S-methionine and its position in the matrix, and the obtained results are illustrated in Fig.6A and 6B. After extraction in vivo-translational reaction mixture with phenol/chloroform and analysis of the products by electrophoresis In LTO-PAG appeared35S-labeled band with the same mobility as mobility matrix 43-R. the Amount of the synthesized material depends on the concentration of Mg2+(Fig.6A). The optimal concentration MD2+is in the range from 9 to 18 mm, which corresponds to the optimal broadcast in the system (Zubay, Ann. Rev. Genet. 7: 267 (1973); Collins, Gene 6: 29 (1979); Chen & Zubay, Methods Enzymol, 101: 44 (1983); Pratt, in reduced and Transiation: A Practical Approach, B. D. Hammes, S. J. Higgins, Eds. (IRL Press, Oxford, 1984), pp. 179-209; and Ellman et al., Methods Enzymol. 202: 301 (1991); Kudlicki et al. (Anal. Chem. 206: 389 (1992)); and Lesley et al. (J. Biol. Chem. 266: 2632 (1991)). In addition, mark has been resistant to treatment NH4OH (Fig.6B), which indicates that this label was localized at the 3'-half molecule (stable with respect to the base DNA) and was joined by resistant base connection, as expected for amide bond between puromycin and fMet.
The dependence on the ribosome and the matrix
In order to demonstrate that the observed higher reaction happens is we (Fig.6C) and the effect of the change sequence, encoding a methionine (Fig.6B). In Fig.6C clearly shows that this reaction is strongly inhibited the peptidyl transferase inhibitors, virginiamycin, goperation and chloramphenicol (Mopho & Vazques, J. Mol. Biol. 28: 161 to 165 (1967); and Vazque & Monro, Biochemica et Biophysical Acta 1423: 155-173 (1967)). In Fig.6D shows that the replacement of one base in the matrix, And With that cancels the inclusion of35S-methionine in 9 mm Mg2+and significantly reduces the number of 18 mm (suggesting that high levels of Mg2+result in incorrect reading of the transcript). These experiments demonstrated that this reaction takes place at the ribosome-dependent matrix method.
The length of the linker
It was also tested the effect of the length of the linker (Fig.6E). This designed the original matrix so that the linker was located at a distance from the decoding site (occupied AUG this matrix) to the acceptor site (employed promicious molecule), where this distance is approximately equal to the distance between anticodon loop and acceptor "stem" in tRNA and is about 60-70 A. First tested the linker had a length of 30 nucleotides at a minimum of 3.4 on A base (102 A). In the range of the efficiency of the reaction. In accordance with the length of the linker may vary. Although the preferred length of the linker is from 21 to 30 nucleotides, in the present invention can also be used linkers, having a length of less than 80 nucleotides, and preferably less than 45 nucleotides.
Intramolecular or intermolecular reactions
Finally, the authors present invention was analyzed whether this intramolecular reaction type (Fig.5, "reaction A"), as it would be desirable, or intermolecular type (Fig.5, "reaction"). This test was performed by adding oligonucleotides with 3'-puromycin, but without sequence binding to the ribosome (i.e. no matrix 25-R, 13-R 30-R), to the translation reaction mixtures containing matrix 43-R (Fig.6F, 6G and 6N). If this reaction occurs by an intermolecular mechanism, it must be marked with shorter oligonucleotides. As shown in Fig.6F-H, in the three shorter oligonucleotides was observed a small inclusion of35S-methionine, which indicates that this reaction occurs mainly on the intramolecular mechanism. The following shows the sequence 25-R (SEQ ID NO:10), 13-R (SEQ ID NO:9) and 30-R (SEQ ID NO:8).
Liz/sup>S-methionine using, in addition to the above lysates of E. coli lysate of rabbit reticulocytes (see below). This reaction, as it preferably is, primarily, by an intramolecular mechanism.
Synthesis and testing of hybrids containing the tag epitope with-ICC
Were also constructed a representative hybrids, which in its protein part contained the epitope-tag for the monoclonal anti-C-ICC antibodies E (Evan et al., Mol. Cell. Biol. 5:3610 (1985)).
The design matrices
Designed three of the original matrix labels for labels-epitopes (i.e LP77, LP154 and Pool #1), which is shown in Fig.7A-C. the First two matrix contained the sequence for the specified with-ICC-epitope tag EQKLISEEDL (SEQ ID NO:2), a third matrix was a design used in the synthesis of a pool for random selection. LP77 encode a sequence of 12 amino acids with codons optimized for bacterial transmission. LP154 and its derivatives contained mRNA sequence encoding a 33 amino acids, where the codons optimized for eukaryotic translation. For the selection of antibodies I used the encoded amino acid sequence MAEEQKLISEEDLLRKRREQKLKHKLEQLRNSCA (SEQ ID NO:7) corresponding to the original peptide. Pool Pool , sootvetstvuyshee the last seven amino acids of the ICC-peptide (which were not part of a series of ICC-epitope). These sequences are shown below.
System in vitro translation using reticulocytes or wheat germ
Matrix 43-R, LP77 and LP154 tested in systems broadcast extracts from rabbit reticulocytes or wheat germ (Promega, Boehringer Mannhiem) (Fig.8). The broadcast was carried out at 30C for 60 minutes. Matrix was isolated using dT25-agarose at 4C. the Matrix was suirable from agarose using a 15 mm NaOH, 1 mm EDTA, neutralized buffer NaOAc/HOAc, immediately precipitated with ethanol (2.5 to about 3.), washed (100% ethanol) and dried in a speed vacuum concentrator. In Fig.8 shows that the35S-methionine was administered in all three matrices, as in wheat germ, and in systems of reticulocytes. Less destruction of the matrix was observed in the fusion reactions obtained in the system of reticulocytes, and why this system is preferred for generating hybrids of RNA-protein". In addition, eukaryotic systems are generally preferred over bacterial systems. Because eukaryotic cells typically contain BH cells. In experiments using a specific system is broadcast E. coli, the formation of hybrids was not observed using matrix encoding with-ICC-epitope; however, the tagging matrix in different areas has demonstrated that it is probably caused by the destruction of the RNA - and DNA-plots of this matrix.
To assess the peptide part of these hybrids, the samples were treated with RNase to remove the coding sequences. After this treatment the product 43-R passed with mobility, almost identical mobility32P-labeled oligonucleotide 30-R, which corresponded to a very small size of the peptide (probably only methionine) attached to a 30-R. if LP77 deleting the coding sequence was produced product with a smaller mobility than the mobility of the oligonucleotide 30-R that correspond to the indication that the peptide of 12 amino acid was attached to puromycin. And finally, in the case LP154, deleting the coding sequence was produced product with even less mobility, which corresponded to the connection sequence of 33 amino acids to oligonucleotide 30-R. On the track reticulocyte LP154, treated with RNase, was not observed in any of the oligonucleotide that is the product, biogas produced in the extract of wheat germ. In General, these results showed that the products that are resistant to the action of RNase were added to the end of oligonucleotides 30-R, the size of these products were proportional to the length of coding sequences, and that these products were absolutely uniform in size. In addition, although both systems have produced similar hybrid products, the system of reticulocytes had the advantage due to the higher stability of the matrix.
Sensitivity to RNase a and proteinase K
As shown in Fig.9, the sensitivity to RNase a and proteinase K was tested with hybrid LP154. As shown in lanes 2-4, the inclusion of35S-methionine was demonstrated for the matrix LP154. When processing this product by RNase And mobility of this hybrid was decreased, but was still significantly higher than the mobility of32P-labeled oligonucleotide 30-R, which corresponded to the accession of the peptide of 33 amino acids to the 3'-end. If this material was also treated with proteinase K, then35S-signal completely disappeared, which is also consistent with the indication that this label was present in the peptide at the 3'end of the fragment 30-R. Similar results would be the merit matrix35S-C was the result of the broadcast, and more specifically, the peptidyl transferase activity of the ribosome, was conducted to estimate the effects of various inhibitors on the response of the tagging. All specific inhibitors of eukaryotic peptidyltransferase, anisomycin, gogarten and sparsomycin (Vazquez, Inhibitors of Protein Biosynthesis (3pringer-Verlag, New York), pp.312 (1979)), and inhibitors of translocation, cycloheximide and emetine (Vazquez, Inhibitors of Protein Biosynthesis (Springer-Verlag, New York), pp.312 (1979)) at ~95% decreased the formation of a hybrid RNA-peptide when using long ICC-matrix and extract broadcast of reticulocytes lysate.
The experiments thus
In the experiment designed to illustrate the efficiency thus hybrid mRNA-peptide, an attempt was made to conduct thus free from-ICC-peptide, generated by in vitro translation. In Fig.10 shows the results of these experiments were analyzed by electrophoresis peptide in SDS page with LTOs. On tracks 1 and 2 shows the labeled material reactions traslochi containing either RNC (RNA-part LP154) or mRNA-globin. On tracks 3-8 shows immunoprecipitate samples of these reactions with the use of monoclonal antibodies is a peptide originating from RNC was effectively immunoprecipitate, while the best case is track 4, where it was allocated ~83% on the entire amount precipitiously TCA. On tracks 6-8 shows a small amount of protein-globin, indicating >100-fold purification. The results showed that the peptide encoded RNC (and LP154), may have quantitative output in accordance with the scheme, thus.
Next, the authors present invention was tested ability to thus chimeric product "RNA-peptide using reactions broadcast LP154 and monoclonal antibodies E against with-ICC (Fig.11). Products broadcast received in the response of reticulocytes, were selected by thus (as described in this application) and treated with 1 mg of RNase And at room temperature for 30 minutes to remove the coding sequence. This allowed us to generate 5 ON, which was marked32P using T4 polynucleotide kinase and analyzed by electrophoresis in denaturing SDS page. In Fig.11 demonstrated that was selected product with a mobility similar to podisi product has not been allocated to broadcast only the RNA portion of this matrix (RNC). In Fig.12 determined the number of allocated hybrid protein and built a graph of the dependence of this quantity on the number of unmodified 30-R (this figure not shown). The quantitative ratio of unmodified linker:hybrid linker-ICC-peptide showed that 0.2 to 0.7% of the built transcript was turned into a hybrid product. A higher fraction of the built-RNA was converted into a hybrid product in the presence of the tested concentrations of mRNA and received approximately 0,8-1,01012the hybrid molecules of one ml translation of the extract.
In addition, the results obtained by the authors showed that the peptides attached to the RNA molecule, were encoded by this mRNA, i.e., given the growing peptide was not transferred to puromycin any other mRNAs. Any evidence of cross-migration was not detected when sharing the incubation linker (30-R) long ICC-matrix in translational extracts for up to 20:1, and was not observed that in the presence of free linker significantly decreased the amount of long produced ICC-hybrid. Similarly, joint broadcast short and long matrices, 43-P and LP154, has produced t is Noah mobility, as expected for hybrids short matrix with long myc-peptide. Both of these results suggest that the formation of the hybrids are mainly between the growing peptide and mRNA associated with the same ribosome.
The subsequent allocation
As an additional confirmation of the nature of the product in vitro-translated matrix LP154 the authors of the present invention conducted an assessment of the behavior of this product on the chromatographic media of two different types. Thiopropyl(TA)-sepharose allows you to select a product containing a free cysteine (for example, L154-product, which has a cysteine residue adjacent to the C-end) (Fig.13). Similarly, dt25the agarose allows you to select a matrix containing poly-da sequence (for example, 30-R) (Fig.13). In Fig.14 demonstrated that, the next selection on the TR-sepharose, and then dt25-agarose received the same product as in the selection only on dt25-agarose. The fact that the product of in vitro translation contained a poly-a-tail and a free thiol, clearly indicates that this product broadcast was a need hybrid RNA-peptide".
The above results point to the possibility of sin part synthesized thus hybrids have the desired sequence, as has been demonstrated by thus and selection using appropriate chromatographic methods. In accordance with the results presented above, these reactions are intramolecular and occur dependent on the matrix method. And finally, even when modifying the matrix is less than 1% of the system of the present invention facilitates the selection based on the complexes of candidates, consisting of about 1013molecules.
The selection with the selection with-ICC-epitopes
For selection of extra-ICC-epitopes generated a large library translational matrices (for example, 1015members), containing randomised region (see Fig.7C and below). This library was used to generate ~1012-1013hybrids (as described in this application), which were treated with antibody against C-ICC (for example, by thus or using antibodies immobilized on a column or other solid media) for enrichment with-ICC-coding matrixes in repeated cycles of in vitro selection.
Models for the formation of hybrids
Without pretending to any particular theory, the authors present invention suggested in relation to the model of the mechanism of formation Gibran. When the ribosome reaches the DNA of this matrix, the transmission is terminated. At this stage, the complex can undergo two processes: dissociation of the growing peptide or the transfer of the growing peptide to puromycin at 3'-end of the matrix. The reaction efficiency of the transfer, probably depends on a number of factors that affect the stability stopped translational complex and embedding of the 3'-promising residue in the a-site of the peptidyl transferase center. After the reaction of transfer of the hybrid RNA-protein", obviously, remains in the form of a complex with the ribosome, since the known factors release cannot hydrolyze stable amide linkage between RNA and peptide domains.
As a classic model for elongation (Watson, Bull. Soc. Chim. Biol. 46: 1399 (1964)) and the model of the intermediate state (Mozald & Noller, Nature 342: 142 (1989)) require that A-site was free for the introduction of puromycin in the peptidyl transferase center. For the implementation of puromycin into the vacant A-site of the linker must either create an outer loop around the ribosome, or to pass directly from the decoding site And the site to the peptidyl transferase center. Described here, the data do not allow for a clear distinction between these alternatives, postovati the ribosome from the outside. In some models the structure of the ribosome (Frank et al., Nature 376: 441 (1995)) mRNA passes through the channel, which extends on either side of the decoding site, and in this case, in order to puromycin reached the peptidyl transferase center through the A-site, it is necessary that this linker is out of the specified channel.
It is obvious that the transfer of the growing chain peptide to puromycin is a slow process compared to the process of elongation, as indicated by the homogeneity and length of the peptide attached to the linker. If during lengthening puromycin effectively competes with the aminoacyl-tRNA, it is expected that hybrids "linker-peptide" present in a hybrid products must be heterogeneous in their size. Moreover, it is clear that the ribosome does not read in the linker region, which is indicated by the similarity of the mobility in the gel hybrid Met-matrix and unmodified linker. da3nshould encode (lysine)nthat should go some way to reduce the mobility of the linker. The low speed output of mRNA can be explained by the low rate of education in comparison with the speed of translocation. Preliminary results suggest that the number formed is ri low temperature, that is probably due to the increased time required to transfer the growing peptide chain to puromycin.
Detailed description of materials and methods
Below is a detailed description of materials and methods related to in vitro-translation and testing hybrids "RNA-protein", including hybrids with ICC-epitope tag.
To generate the above hybrids "RNA-protein" was used a number of oligonucleotides. These oligonucleotides had the following sequences.
All of these oligonucleotides are given in the direction of 5’3’. Ribonucleotidic grounds indicated in lowercase letters "r" before the letters nucleotide; R means puromycin; rN denotes equal amounts of ha, rG, rC, and rU; rS indicates an equal number rG and rC; and all other designations grounds indicate DNA oligonucleotides.
Puromycin, Hcl, glass with adjustable long-alkylamino pore size, gogarten, chloramphenicol, virginiamycin, DMAP, dimethyltrimethylene and acetic anhydride were obtained from the company Sigma Chemical (St. Louis, MO)al (Ronkonkoma, NY). the mRNA of beta-globin was obtained from Novagen (Madison, WI). TMV RNA was obtained from Boehringer Mannhiem (Indianapolis, IN).
Proteinase K was obtained from Promega (Madison, WI). Mcasa without Gnkazy, was either obtained in accordance with the Protocol described by Sambruna and others (see above), or purchased from Boehringer Mannheim. The T7 polymerase was obtained in accordance with the Protocol published Grodberg & Dunn (J. Bacteriol. 170: 1245 (1988)), with modifications Zawadski & Gross (Nucl. Acids. Res. 19: 1948 (1991)). DNA T4 ligase were obtained from New England Biolabs (Beverly, MA).
Quantitative assessment include radioactive labels
For bands of the gel with a radioactive label number present radioactive label (35S or32P) in each band was determined by a quantitative or using blot analyzer Betagen 603 (Betagen, Waltham, MA), or using a phosphor imaging plates (Molecular Dynamics, Sunnyvale, CA). For liquid and solid samples, the number present radioactive label (35S or32P) was determined by counting in a scintillation counter (Beckman, Columbia, MD).
Gels were visualized by autoradiography (using film Kodak XAR) or using a phosphor imaging plates (Molecular Dynamics).
Synthesis of CPG-vs acid for holding kinase reactions, reactions of transcription, PCR and reactions broadcast using extracts from E. coli was obtained in the same way. Each preparative Protocol began with extraction with equal volume of phenol/chloroform, 1:1, and then spent centrifugation and isolation of the aqueous phase. Sodium acetate (pH 5,2) and spermidine was added to a final concentration of 300 mm and 1 mm, respectively, and the sample was besieged by adding 3 volumes of 100% ethanol and incubation at -70C for 20 minutes. Then the samples were centrifuged at >12000g, the supernatant was removed and the precipitate was washed with excess of 95% ethanol at 0C. then the precipitate was dried in vacuum and resuspendable.
All synthetic DNA and RNA synthesized in the synthesizer Millipore Expedite standard methods of chemical synthesis for each of them in accordance with the manufacturers recommendation (Milligen, Bedford, MA). Oligonucleotides containing 3’-puromycin, was synthesized on columns with CPG-puromycin, Packed 30-50 mg of solid media (~20 µmol puromycin-on/gram). Oligonucleotides containing 3'-Biotin was synthesized on columns using 1 mmol of biomedi CPG from Glen Research (Sterling, VA). Oligonucleotides, sodalykakugy, lagerwey with 3'-ends of RNA molecules or chemically was fosforilirovanii the 5'-end (using chemical fosforiliruyusciye reagents from Glen Research) before release, or enzymatic were fosforilirovanii using ATP and T4 polynucleotide kinase (New England Biolabs) after release. Samples containing only DNA (3'-puromycin or 3'-Biotin) were subjected to release by adding 25% of NH4OH, followed by incubation for 12 hours at 55C. Samples containing RNA monomers (e.g., 43-R), were released by the addition of ethanol (25% vol./about.) to a solution of NH4OH, and then incubated for 12 hours at 55C. 2 ON was unblocked using 1 M TBAF in THF (Sigma) for 48 hours at room temperature. TBAF was removed using a column with Sephadex NAP-25 (Pharmacia, Piscataway, NJ).
If necessary, for the implementation of the test for the presence of 3'-hydroxyl group puromycin-oligonucleotide may be radioactively labeled at the 5'-end using T4 polynucleotide kinase, and then used as a primer for extension terminal deoxynucleotidyltransferase. The presence of the primary amine in puromycin can be p the Chida, such as 30-R, find detective change in mobility during electrophoresis in denaturing SDS page, conducted after the reaction, indicating a quantitative reaction with this reagent. Oligonucleotides that do not contain puromycin, do not react with NHS-LC-Biotin and do not detect changes in the mobility.
Then the released DNA and RNA samples were purified using electrophoresis in denaturing SDS page, and then subjected to either wetting or electroelution from the gel using an eluting traps Elutrap (Schleicher & Schuell, Keene, NH) and desalting using either column with Sephadex NAP-25, or precipitation with ethanol as described above.
The construction of the ICC DNA
Were designed two DNA template containing a tag with-ICC-epitope. The first matrix was created from the combination of oligonucleotides 64.27 (5'-GTT CAG GTC TTC YYG AGA GAT CAG TTT CTG TTC CAT TTC GTC CTC CCT ATA GTG AGT CGT ATT A-3') (SEQ ID NO:18) and 18.109 (5'-TAA TAC GAC TCA HUNDRED TAG-3') (SEQ ID NO:19). The resulting transcription using this matrix was producirovanie RNA 47.1, which encodes the peptide MEQKLISEEDLN (SEQ ID NO:20). After ligating RNA 47.1 30-R received LP77, shown in Fig.7A.
The second matrix is first produced in the form of one of the oligonucleotide length in 99 reasons indicated RWR 99.6 ID NO:21). Double-stranded transcription matrix containing this sequence was constructed by PCR using oligonucleotides RWR 21.103 (5'- AGC GCA AGA GTT AGC CAG CTG-3') (SEQ ID NO:22) and RWR 63.26 (5'- TAA TAC GAC TCA HUNDRED TAG GGA CAA TTA HUNDRED TTT ACA ATT ACA ATG GCT GAA GAA CAG AAA CTG -3') (SEQ ID NO:23) in accordance with published protocols (Ausubel et al., see above, Chapter 15). The resulting transcription using this matrix was obtained RNA, which meant RNA 124 and which encodes a peptide:
MAEEQKLISEEDLLRKRREQLKHKLEQKRNSCA (SEQ ID NO:24). This peptide contains the sequence used to generate the monoclonal antibodies E during conjugation with protein carrier (Oncogene Science Technical Bulletin). RNA 124 had a length of 124 nucleotide, and after ligating RNC with 30-R received LP154, shown in Fig.7V. The RNA sequence 124 is a (SEQ ID NO:32):
5'-rGrGrG rArCrA rArUrU rArCrU rArUrU rUrArC rArArU rUrArC rArArUrG rGrCrU rGrArA rGrArA rCrArG rArArA rCrUrG rArUrC rUrCrU rGrArA rGrArC rCrUrG rGrarC rCrUrG rCrUrG rArArA rCrGrU rCrGrU rGrArA rCrArG rCrUrG rArArA rCrArC rArArA rCrUrG rGrAra rCrArG rCrUrG rCrGrU rArArC rUrCrU rUrGrC rGrCrU-3'
Design randomized pool
Randomized pool designed in the form of one nucleotide in length 130 bases, marked RWR 130.1. This sequence is, in the direction from the 3'-end 3'-CCCTGTTAATGATAAATGTTAATGTTAC (NN the van in accordance with the standard scheme of synthesis. S means equal mixture of bases dG and dC. PCR was performed using oligonucleotide 42.108 (5'-TAA TAC GAC TCA HUNDRED TAG GGA CAA TTA HUNDRED TTT ACA ATT ACA) (SEQ ID NO:26) and 21.103 (5'-AGC GCA AGA GTT ACG CAG CTG) (SEQ ID NO:27). In the transcription of this matrix was obtained RNA, labeled Pool 130.1. After ligating the Pool 130.1 30-R received l #1 (also denoted LP160), shown in Fig.7C.
Seven cycles of PCR were carried out according to published protocols (Ausubel et al., see above), except that (i) the initial concentration RWR 130.1 was 30 nanomoles (ii) each primer was used at a concentration of 1.5 μm, (iii) the dNTP concentration was 400 μm for each base, and (iv) Taq polymerase (Boehringer Mannhiem) was used at a concentration of 5 units per 100 μl. Double-stranded product was purified by electrophoresis in sedentarism PAG and allocated by electroelution. The amount of DNA was determined by UV absorption at 260 nm, and compared ethidiumbromid fluorescence with known standards.
Enzymatic RNA synthesis
Response transcription of double-stranded PCR DNA and sinteticheskih oligonucleotides was performed as described previously (Milligan and Uhlenbeck, Meth. Enzymol. 180: 51 (1989)). Full-RNA was purified by electrophoresis in denaturing SDS page,into extinction 1300 OD/μmol; RNA 124, 1250 OD/μmol; RNA 47.1, 480 OD/μmol. In the transcription of double-stranded DNA pool got ~90 nanomoles RNA pool.
Enzymatic synthesis of conjugates RNA puromycin Ligation of sequences of matrix ICC-RNA and RNA pool with puromycin containing oligonucleotide was performed using DNA-shunt marked 19.35 (5'-TTT TTT TTT TAG CGC AAG (A) (SEQ ID NO:28) using a procedure analogous to the procedure described by Moore & Sharp (Science 250: 992 (1992)). The reaction mixture consisted of mRNA, "shunt" and puromycin-oligonucleotide (30-R, dA27dCdCP) in a molar ratio 0,8:0,9:1,0, 1-2,5 units of DNA ligase at one picomole mRNA pool. The reaction was carried out for one hour at room temperature. To construct hybrids RNA pool, the concentration of mRNA was ~6.6 µmol. After ligation, the conjugate RNA puromycin received as described above for enzymatic reactions. The precipitate resuspendable, and a full-sized hybrids were purified by electrophoresis on denaturing SDS page, and identified by electroelution as described above. The RNA pool was estimated using the extinction coefficient 1650 OD/μmol, and the ICC-matrix - 1600 OD/μmol. Thus, there was obtained 2.5 nanomoles conjugate.
Receipt is Noah biological label (Glen Reasearch) and desalted on a column (NAP-25 (Pharmacia)) were incubated at a concentration of 1-10 μm, or even 1-20 microns with a suspension of streptavidin-agarose (50% vol. agarose, Pierce, Rockford, IL) for 1 hour at room temperature in TE (10 mm Tris-chloride, pH of 8.2, 1 mm EDTA) and washed. Then binding capacity of agarose was evaluated optically by the disappearance of the Biotin-dt25from a solution and/or by titration of the resin with known quantities of complementary oligonucleotide.
Reactions broadcast using extracts and ribosomes occurring E. coli
Basically, reactions broadcast was performed using a purchased sets (for example, a set of E. coli S30 Exstract for Linear Templates, Promega, Madison, WI). However, to generate S30 extracts obtained in accordance with published protocols (for example, Ellman et al., Meth. Enzymol. 202: 301 (1991)), and ribosomal fractions, obtained as described Kudlicki et al. (Anal. Chem. 206: 389 (1992)) can also be used a strain of E. coli MRE600 (obtained from ATS, Rockville, MD). The standard reaction was carried out in 50 µl-volume using 20-40 µci35S as a marker. Reacciona mixture consisted of a mixture of 30% (vol./about.) extract, 9-18 mm MgCl2, 40% (vol./about.) premix containing no methionine (Promega), and 5 μm of the matrix (e.g., 43-R). For the experiments on co-incubation of the oligonucleotide 13-P 25-P was added at a concentration of 5 μm. For experiments using ribosomes �D/chr/176.gif">C for 30 minutes. Matrix was purified as described above in enzyme reactions.
Reactions translation using wheat germ
Reactions broadcast, shown in Fig.8, was performed using commercially available kits that do not contain methionine (Promega) according to manufacturers instructions. Concentration matrix was 4 μm to 43-P and 0.8 µm for LP77 and LP154. The reaction was carried out at 25With using 30 µci35S-methionine in the full volume of 25 µl.
Reactions broadcast using reticulocytes
Reactions broadcast was carried out either using commercially available kits (Novagen, Madison, WI), or by use of the extract obtained in accordance with published protocols (Jackson & Hunt, Meth. Enzymol. 96: 50 (1983)). Enriched with reticulocytes blood was obtained from Pel-Freez Biologicals (Rogers, AK). In both cases, the reaction conditions were the conditions recommended for use with lysate Red Nova Lysate (Novagen). The reaction mixture consisted of 100 mm KCl, 0.5 mm, MDAs, 2 mm DTT, 20 mm HEPES, pH of 7.6, 8 mm creatine phosphate, 25 μm of each amino acid except methionine, if you use35S-Met), and 40% (vol./about.) lysate. Incubation was carried out at 30To generate the randomized pool response broadcast in a volume of 10 ml was carried out at a concentration of matrix ~0.1 mm (1.25 nanomoles matrix). In addition,32P-labeled matrix included in the reaction to determine the amount of material present in each stage of the purification procedure and selection. After the broadcast at 30C for 1 hour, the reaction mixture was cooled on ice for 30-60 minutes.
The selection of the hybrid using dt25-streptavidin-agarose or oligo dT-cellulose
After incubating the reaction mixture for broadcast approximately 150-fold diluted buffer allocation (1.0 M NaCl, 0.1 M Tris-chloride, pH of 8.2, 10 mm EDTA, and either 1 mm DTT, or 0.2% of Triton X-100) containing more than 10x molar excess dt25-Biotin-streptavidin-agarose, where the concentration of dT25was ~10 μm (volume of suspension is equal to or exceeds the amount of lysate), or oligo-dT-cellulose (Pharmacia), and incubated under stirring at 4C for one hour. Then the agarose was removed from the mixture or by filtration (Millipore ultrafree MC filters) or by centrifugation and washed 2-4 times cold bumioc 50-100 µl-aliquot of 15 mm NaOH, 1 mm EDTA at 4With or pure water at room temperature. Eluent was immediately neutralized in 3 M Na, pH of 5.2, 10 mm spermidine and precipitated with ethanol or used directly for the next stage of purification. For the reaction pool total released radioactivity was approximately 50-70% of the allocated enabled matrix.
The selection of the hybrid using thiopropyl-sepharose
Hybrids containing cysteine, can be cleaned using dipropylacetate 6B, as shown in Fig.13 (Pharmacia). In the experiments described in this application, the selection is carried out either directly by the reaction of a broadcast, or after the initial selection of the hybrid (e.g., using streptavidin-agarose). For the samples treated by the direct method, the ratio of the lysate to sepharose was 1:10 (vol./vol.). For the pool 0.5 ml suspension sepharose used to select the entire hybrid material from 5 ml of the reaction mixture. Samples were diluted suspension of 50:50 (vol./about.) dipropylacetate in 1 x TE 8,2 (10 mm Tris-Cl, 1 mm EDTA, pH 8,2) containing RNase without Gnkazy (Boehringer Mannhiem) and incubated with stirring rotation for 1-2 h at 4For completion of the reaction. The excess liquid is a Finance or filtering. Hybrids were suirable of sepharose using solution 25-30 mm dithiotreitol (DTT) in 10 mm Tris-chloride, pH of 8.2, 1 mm EDTA. Then the hybrid concentrated by combining evaporation in high vacuum, precipitation with ethanol, as described above, and, if necessary, and analyzed by electrophoresis in LTO-Trizin-PAG. For the reaction in the pool total released radioactivity showed that approximately 1% of the matrix was turned into a hybrid.
In some cases, dT25added to this eluate was mixed by rotation for 1 hour at 4C. the Agarose was washed three times with cold buffer for selection were isolated by filtration, and the bound material was suirable as described above. Then add the carrier tRNA, and hybrid product precipitated with ethanol. Sample resuspendable in TE, pH of 8.2, containing RNase And without Gnkazy to remove the RNA part of the matrix.
The reaction thus
Immunoprecipitation peptides from the reactions of translation (Fig.10) was made by mixing 4 μl translation reaction mixture containing reticulocytes, 2 μl of normal mouse serum and 20 μl of protein G plus agarose A (Calbiochem, La Jolla, CA), 200 μl of either PBS (58 mm Na2HPO4, 17 mm NaH2PO4, 68 mm NaCl), is desoxycholate, 0.1% of LTOs). Then the samples were mixed by rotation for one hour at 4C, and then centrifuged at 2500 rpm for 15 minutes. Eluent was removed and added to 10 ál of monoclonal antibodies E against with-ICC (Calbiochem, La Jolla, CA) and 15 μl of protein G plus agarose And then the mixture was mixed by rotation for 2 hours at 4C. Then the samples were washed with two 1 ml of either PBS or buffer for cultivation or PBSSTDS. To the mixture was added 40 μl of buffer for loading gel (Calbiochem Product Bulletin), and 20 μl was loaded on a denaturing SDS page as described Shagger & von Jagow (Anal. Biochem. 166: 368 (1987)).
Immunoprecipitation hybrids (as shown in Fig.11) was carried out by mixing 8 μl translation reaction mixture containing reticulocytes, with 300 ál of dilution buffer (10 mm Tris-chloride, pH of 8.2, 140 mm NaCl, 1% vol./about. Triton X-100), 15 μl of protein G-sepharose (Sigma) and 10 μl (1 μg) antibody E against c-myc (Calbiochem), followed by stirring rotation for several hours at 4C. After extraction, the samples were washed, treated with RNase And without Gnkazy, were labeled using polynucleotide kinase and32P-gamma-ATP and separated by electrophoresis in denaturing SDS page is accordance with the manufacturer's recommendations for Superscript II, except that the matrix, water, and primer were incubated at 70For only two minutes (Gibco BRL, Grand Island, NY). For monitoring lengthening in some reactions included 50 µci alpha32R-d; in other reactions monitoring reverse transcription was performed using 5'-32P-labeled primers, which were obtained using32P-ATP (New England Nuclear, Boston, MA) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA).
Getting protein G and antibody-sepharose
Two aliquots of 50 μl of the suspension conjugate "G protein-sepharose" (50% vol. solids) (Sigma) were washed with buffer for cultivation (10 mm Tris-chloride, pH of 8.2, 140 mm NaCl, 0.025% Of NaN3, 1% vol./about. Triton X-100) and were isolated by centrifugation. The first aliquot was retained for use in predalone before selection matrix. After resuspendable second aliquot buffer for dilution was added to 40 μg of the monoclonal antibody AB-1 against c-myc (Oncogene Science) and the reaction mixture was incubated overnight at 4With stirring by rotation. Then, the complex of antibody-sepharose was cleared by centrifugation for 15 minutes at 1500-2500 rpm in microcentrifuge and washed 1-2 times by reverse transcriptase was used directly in the selection process. This application describes two protocols. For the first cycle of the reaction mixture with reverse transcriptase was added directly to the complex antibody-sepharose" obtained as described above and incubated for 2 hours. For the subsequent cycles before use speakers with the antibody, the reaction mixture was incubated for ~2 hours and washed conjugate protein G-sepharose" to reduce the number of binders that interact with protein G and not to the immobilized antibody.
Elution of the pool from the matrix can be accomplished by several methods. In the first method, carried out the washing of the matrix for selection of 4% acetic acid. This procedure leads to the release of the peptide from matrix. Alternatively, instead of or in addition to the specified method with the use of acetic acid can be made more stringent washing (for example, with the use of urea or other denaturing substances).
PCR of selected hybrids
Selected molecules are amplified using PCR according to standard protocols (for example, Fitzwater & Polisky, Meth. Enzymol. 267: 275 (1996); and Conrad et al., Meth. Enzymol. 267: 336 (1996) described above to construct the pool. To ensure that this amplification PCR control. The most important factor is the purity of the primer. Couples should be amplified in the absence of the built-in matrix due to the fact that sometimes contamination may occur pool sequences or control structures. When contamination is detected must be synthesized new primers. To ensure that these dedicated hybrids will not be able CDNA impurities before stage RT they should be subjected to stage PCR. And finally, there should be a comparison of the number of cycles required for the implementation of PCR before and after sampling. A large number of cycles required for amplification of the sequence (>25-30 cycles of PCR), may indicate an unsuccessful response RT, or the difficulties associated with the pairs of primers.
Synthesis and testing of hybrids beta-globin
For the synthesis of hybrid structures-globin cDNA-globin generated from 2.5 mg of globin mRNA by reverse transcription reaction with 200 Ptolemy primer 18.155 (5' GTG GTA TTT GTG AGC CAG) (SEQ ID NO:29) and reverse transcriptase Superscript (Gibco BRL) according to the Protocol recommended by the manufacturer. This Primera sequence was complementary to 18 nucleotidesLa reverse transcription was removed and subjected to 6 cycles of PCR using primers 18.155 and 40.54 (5' TAA TAC GAC TCA HUNDRED TAG GGA CAC TTG CTT TTG ACA CAA) (SEQ ID NO:30). Then the obtained mRNA "blue--globin" generated by unregulated transcription T7, as described Milligan & Uhlenbeck (Methods Enzymol. 180: 51 (1989)), and this RNA was subjected to gel purification was electrolytically and absoluely, as described in this application. Then generate "L-globin" from design "blue--globin" by ligating this design with 30-R method, described Moore & Sharp (Science 256: 992 (1992)) using primers 20.262 (5' TTT TTT TTT T GTG GTA TTT G) (SEQ ID NO:31) as a shunt. Then, the reaction product of ligation was subjected to gel purification was electrolytically and absoluely, as described above. The concentration of the final product was determined by optical density at 260 nm.
These matrices-globin aired in vitro, as described in table 1, in total volume of 25 μl each. Then added MD2+25 mm initial solution. All reaction mixtures were incubated at 30C for one hour and left overnight at -20C. Then double-determined number of them/min deposited dT25using 6 μl of lysate was averaged with vietanam background.
To sample the 100 mm Tris-Cl, pH of 8.2, 10 mm EDTA, 0.1 mm DTT), 1 ál of RNase A (without Gnkazy, Boehringer Mannhiem) and 20 μl of 20 μm dt25-streptavidin-agarose. Samples were incubated at 4C for one hour with stirring by rotation. Excess buffer allocation was removed and the samples were applied to the filter Millipore MC in order to remove any remaining buffer to allocate. Then the samples four times washed with 50 µl of N2O and twice with 50 μl of 15 mm NaOH, 1 mm EDTA. A sample (300 µl) was neutralized with 100 ál of TE, pH 6.8 (10 mm Tris-Cl, 1 mm EDTA) was added 1 μl of 1 mg/ml RNase A (as described above), and the samples were incubated at 37C. Then was added 10 μl of 2 x LTO-buffer to load (125 mm Tris-Cl, pH 6.8, 2% LTOs, 2%-mercaptoethanol, 20% glycerol, about 0.001% bromophenol blue), and the sample was liofilizovane dry and resuspendable in 20 ml of N2O and 1%-mercaptoethanol. After that, the samples were loaded on peptideatlas gel described by Schagger & von Jagow (Analitical Biochemistry 166: 368 (1987)) and visualized using autoradiography.
The results of these experiments are presented in Fig.15A and 15C. As shown in Fig.15A,35S-methionine was incorporated into the protein of hybrids "blue--globin" and "LP--globin. In addition, as shown in Fig.15V, after highlighting dT25and cleavage by RNase And on the tracks Shin--globin not remained35S-labeled material (Fig.15V, lanes 2-4). In contrast, on the tracks LP--globin was observed homogeneity of dimensions35S-labeled product.
These results indicate that, as mentioned above, a hybrid product was separated using affinity chromatography of oligonucleotides, only when this matrix contained the 3'-puromycin. This was confirmed by scintillation counting (see table 1). As expected, the resulting material contained 30-R-linker, merged with a part-globin. Hybrid product was absolutely homogeneous in size, as it was shown by gel analysis. However, since this product was found mobility, almost similar to the natural mobility-globin (Fig.15A and 15 B, the control track), it was difficult to determine the exact length of the protein part of the hybrid product.
Additional optimization of the formation of a hybrid RNA-protein"
It was found that some factors potusa peptide chain from its tRNA to promicious part of the 3'-end of mRNA, is a slow reaction that occurs after the initial relatively rapid translation of open reading frames by generating a growing peptide chain. The degree hybrid can be significantly increased by post-translational incubation in conditions of increased MD2+preferably, in the range of 50-100 mm) and/or by using a more flexible linker between mRNA - and promicious part. In addition, prolonged incubation (12-48 hours) at low temperatures (preferably, -20C) also leads to an increase in yield hybrids with less destruction of mRNA than those who receive during incubation at 30C. With a combination of these factors up to 40% of mRNA can be converted into hybrid products "mRNA-peptide", as shown below.
Synthesis of conjugates of mRNA-puromycin
In these experiments, optimization parametersetname linker ligated with 3'-ends of mRNA using the bacteriophage DNA T4 ligase in the presence of complementary DNA shunts, basically as described above. Because DNA ligase T4 prefers the exact base pairing near the junction ligation and products of the samples (Nucleic Acids Research 15: 8783 (1987)), they were effectively legirovanyh only those RNAS that contain the correct 3'-terminal nucleotide. When using standard DNA shunt approximately 40% of the products are unregulated transcription were legirovanyh with puromycin-oligo. The number of very product increased when using redundant RNA, but it did not increase when using redundant puromycin-oligo. Without pretending to any particular theory, it is possible to say that the limiting factor for ligation is the amount of RNA that is fully complementary to the corresponding region of DNA shunt.
For ligating of those transcripts, which ended more nematicidal the nucleotide at the 3'-end (called "N+1-products"), used a mixture of standard DNA shunt with the new DNA-shunt containing additional random basis at the junction ligation. The efficiency of ligation was increased by more than 70% for typical ICC-RNA-matrix (that is, RNA 124) in the presence of the specified mixed DNA shunt.
In addition to the modified method using DNA shunt efficiency education conjugate mRNA-puromycin can be further optimized by considering the following three faktorov significant, stable secondary structure, which is supposed to prevent annealing of the oligonucleotide to the shunt. In addition, since a high concentration of salt sometimes leads to inefficiency in the ligation reaction, this procedure is preferable to stage a thorough desalting of oligonucleotides using columns NAP-25. And finally, because this ligation reaction proceeds relatively quickly and mostly ends in 40 minutes at room temperature, it is usually not used for longer periods of incubation, which often lead to nezhelatelno destruction of RNA.
Using the above conditions compared to mRNA-puromycin were synthesized as follows. Ligation of sequences ICC-RNA (RNC) proministries the oligonucleotide was performed using either a standard DNA-shunt (for example, 5'-TTTTTTTTTTAGCGCAAGA) (SEQ ID NO:32), or shunt, containing an arbitrary base (N) at the junction ligation (for example, 5'-TTTTTTTTTTAGCGCAAGA) (SEQ ID NO:33). The reaction mixture consisted of mRNA, DNA-shunt and puromycin-oligonucleotide in a molar ratio of 1.0:1.5 to 2.0:1.0 in. Can also be used an alternative molar ratio of 1.0:1,2:1,4. First, the mixture of these component of religiouse was carried out for one hour at room temperature in 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 10 mm DTT, 1 mm ATP, 25 μg/ml BSA, 15 μm puromycin-oligonucleotide, 15 μm mRNA, 22.5 to 30 μm DNA-shunt, an inhibitor of Mcsina (Promega) at a concentration of 1 unit/ál and 1.6-2.5 units DNA ligase T4 on one picomole puromycin-oligonucleotide. After incubation were added EDTA to a final concentration of 30 mm, and the reaction mixture was extracted with a mixture of phenol/chloroform. Full conjugates were purified by electrophoresis in denaturing SDS page, were isolated by elektrobudowa and absoluely.
General conditions broadcast using reticulocytes
In addition to improving the efficiency of synthesis of conjugate "mRNA-puromycin", reactions broadcast were also optimized in the following way. The reaction was carried out in the lysate of rabbit reticulocytes obtained from various commercial sources (Novagen, Madison, WI; Amersham, Arlington Heights, IL; Boehringer Mannhiem, Indianopolis, IN; Ambion, Austin, TX; and Promega, Madison, WI)). A typical reaction mixture (final volume 25 µl) consisted of 20 mm HEPES, pH of 7.6, 2 mm DTT, 8 mm creatine phosphate, 100 mm KCl, 0.75 mm MD (SLA)2, 1 mm ATP, 0.2 mm GTP, 25 μm of each amino acid (0,7 µm methionine, if you use35S-Met), Rcasino at a concentration of 1 unit/μl and 60% (vol./about.) lysate. The final concentration of the matrix was in the range from 50 nm to 800 Ni lysate was thawed immediately before use. After adding the lysate reaction mixture was carefully mixed by gentle pipetting and incubated at 30For initiation of translation. For different mRNA optimal concentrations MD2+and+ranged from 0.25 mm to 2 mm and 75 mm to 200 mm, respectively, and preferred concentrations were determined in preliminary experiments. In particular, for poorly translated mRNAs sometimes also optimized concentration of hemin, creatine phosphate, tRNA and amino acids. For reactions with the formation of hybrids of the potassium chloride was mostly preferred over potassium acetate, and a mixture of KCl and COAs sometimes gave the best results.
After the broadcast at 30From within 30-90 minutes the reaction mixture was cooled on ice for 40 minutes and was added Mg2+or2+. At this stage the final concentration MD2+optimized for different mRNA-matrices, and this concentration was in the range from 50 mm to 100 mm (for pools of mixed matrices preferably used 50 mm). The number added To the2+usually was in the range from 125 mm to 1.5 M For the reactions of Mg2+the resulting mixture is preferably inkaso. If added To the+or MD2+/K+then the mixture is incubated at room temperature for one hour.
To visualize the labeled hybrid products 2 μl of reaction mixture was mixed with 4 μl of the boot buffer, and the mixture was heated at 75C for 3 minutes. Then this mixture was loaded onto LTO-polyacrylamide gel containing 6% glycine (32P-Met-labeled matrices) or LTO-polyacrylamide gel containing 8% Trizin (35S-Met-labeled matrices). As an alternative to this method, hybrid products can also be selected using dt25-streptavidin-agarose or dipropyl-sepharose (or both), mainly as described in this application.
To remove the RNA portion of the RNA conjugate-linker-puromycin-peptide for subsequent analyses using elecrophoresis in LTO-SDS page after carrying out post-translational incubation was added the appropriate amount of EDTA, and the reaction mixture was absoluely using column Microcon-10 (or Microcon-30). 2 μl of the mixture (approximately 25 ml) was mixed with 18 μl of buffer RNase H (30 mm Tris-HCl, pH of 7.8, 30 mm (NH4)2SO4, 8 mm MgCl21.5 mm-mercaptoethanol and soul for 45 minutes. Then add the RNase H cleavage was carried out at 37C for 20 minutes.
The number of puromycin-oligonucleotide
The number of puromycin-oligonucleotide is also important for the efficient production of hybrid products. The binding of 5'-DMT-, 2'-succinyl-, N-TRIFLUOROACETYL-puromycin with CPG was not as effective as the standard binding of nucleotides. When it carried out a thorough monitoring of the binding assays to avoid the formation of CPG too low concentration associated puromycin, and unreacted amino groups on the CPG were completely inactivated in order to avoid subsequent synthesis of oligonucleotides containing 3'-terminal puromycin. It is also important to avoid the use of CPG containing very small particles that can lead to the formation of lumps in the valves during the subsequent stages of the automated synthesis of oligonucleotides.
In addition, before large-scale use of synthesized puromycin-oligonucleotide preferably tested to ensure the presence of puromycin at the 3'-end. In experiments conducted by the authors of the present invention, the hybrid was not found in case pctia 3'-terminal hydroxyl groups (i.e. unwanted synthesis of oligonucleotides, does not contain a 3'-terminal puromycin), this puromycin-oligonucleotide may be first radioactively labeled (for example, 5'-phosphorylation), and then used as a primer for elongation under the action limit deoxynucleotides-ferazi. In the presence of the 3'end promicious part should not be lengthening product.
Time for broadcast and post-translational incubation
The reaction of the broadcast was relatively rapid and was mostly completed in 25 minutes at 30C. However, the reaction of formation of the hybrid was slower. When using the standard linker (dA27dCdCP) if 30With the fusion hybrid has reached its maximum level in the next 45 minutes. Posttranscriptional incubation can be carried out at lower temperatures, for example at room temperature, 0C or at -20C. Less destruction of the mRNA-matrix was observed at -20And the best educational outcomes hybrids were obtained after incubation at -20With in 2 days.
The effect of the concentration MD2+or+
High of hybrid. For example, ICC-RNA-matrix described above, 3-4-fold stimulation of the formation of the hybrid was observed using the standard linker (dA27dCdCP) in the presence of 50 mm Mg2+during the 16-hour incubation at -20With (Fig.17, compare lanes 3 and 4). Effective education hybrid was also observed with the use of post-translational incubation in the presence of a concentration of 50-100 mm MD2+if the data reaction was carried out at room temperature for 30-45 minutes. Similarly, adding 250-500 mm+led to a more than 7-fold increased education of the hybrid compared to the control, which was not added To the+. The optimal concentration To+usually ranged from 300 mm to 600 mm (500 mm for pools). Post-translational addition of NH4Cl has also led to increased formation of the hybrid. The choice SLA-or CL-as the anion does not have a significant impact on the formation of the hybrid.
The length and sequence of the linker
It was also evaluated the dependence of the reaction of formation of the hybrid on the length of the linker. Within 21-30 nucleotides (n=18-27), there was slight change in the efficiency of the reaction of formation of hybrid (as described by wyssbrod was observed for linkers of 25 nucleotides (Fig.23). Shorter linkers (e.g., 13 or 16 nucleotides in length) or longer linkers (e.g., linkers, with more than 40 nucleotides in length) gave a much lower level of education hybrids. In addition, although the use of specific linkers with greater length (i.e. from 45 and 54 nucleotides of nucleotides) also led to some extent to the decrease in the efficiency of formation of the hybrid, however, to optimize the efficiency of the reaction of formation of the hybrid, probably, can be used even longer linkers.
As for the linker sequence, replacing deoxy-ribonucleotidic residue near the 3'-end on ribonucleotidic residues does not lead to a significant change in the efficiency of the hybrid. However, the sequence dCdCP (or gsgs) at 3'-end of the linker is important for the formation of the hybrid. Replacement dCdCP on dUdUP leads to a significant reduction efficiency of a hybrid.
The flexibility of the linker
It was also tested the dependence of the reaction of formation of the hybrid from the flexibility of the linker. In these experiments it was determined that the efficiency of formation of the hybrid was low in the case when the stiffness was increased by annealing with complC9C9C9dAdCdCP, where C9is BUT (CH2CH2O)3RHO2), the effectiveness of education hybrid was significantly increased. Compared with the standard linker (dA27dCdCP) use more rigid linker (dA21C9C9C9dAdCdCP) contributes to the effectiveness of education hybrid for RNC more than 4 times (Fig.17, compare lanes 1 and 9). In addition, unlike the matrix with the standard linker, posttranslational hybrid which is produced by poorly in the absence of high concentrations MD2+(Fig.17, lanes 3 and 4), the matrix with a flexible linker does not require an increase in the concentrations MD2+for producing a good output hybrid product with prolonged post-translational incubation at -20With (Fig.17, compare lanes 11 and 12). Therefore, this linker was most appropriate in that case was not required to conduct post-translational additions of high concentrations of Mg2+. In addition, this hard-linker was also possible to produce a hybrid with the best outputs in the presence of increased concentrations of Mg2+.
Quantitative evaluation of the effectiveness of hybrid
Effective product either as a fraction of the built-in matrix, transformed into a hybrid product. To determine the fraction of the translated peptide, turned into a hybrid product, this translated peptide were labeled35S-Met. In these experiments, where we used the linker dA27dCdCP or dA27rCrCP, after 1-hour translational incubation at 30With approximately 3.5% of the translated peptide was hybridized with its mRNA. After incubation overnight at -20With this value increased to 12%. If post-translational incubation was carried out in the presence of high concentrations of Mg2+then the matrix was hybridisable more than 50% translational peptide.
For a matrix with a rigid linker after 1-hour broadcast at 30With this matrix was hybridisable about 25% of the translated peptide. This value was increased by more than 50% after an overnight incubation at -20With more than 75% in the case where post-translational incubation was performed in the presence of 50 mm MD2+.
To determine the percentage of transformation of the built-in matrix in a hybrid product, the broadcast was carried out with ispolizovanie was carried out at -20With no added MD2+approximately 20%, 40%, 40%, 35% and 20% of the built-in matrix was converted into a hybrid mRNA-peptide, where the integrated concentration of the RNA matrix was 800, 400, 200, 100 and 50 nm, respectively (Fig.18). Similar results were obtained if the post-translational incubation was carried out in the presence of 50 mm MD2+. The best results were achieved using lysates obtained from Novagen, Amersham or Ambion (Fig.19).
If this mRNA-matrix is long, the differences in mobility between mRNA and hybrids of mRNA-peptide defined by electrophoresis in SDS page with LTOs can be very small. In such cases, this matrix can be32P-labeled at the 5'-end linker (for example, with the use of [32R]ATP and Poliny-leticias T4 with subsequent legirovaniem with conjugate mRNA-puromycin). Then, after the broadcast/incubation, long RNA portion can be cleaved by RNase H in the presence of complementary DNA shunt, and the effectiveness of this hybrid is determined by a quantitative assessment of the relationship unmodified linker to the hybrid linker-peptide. Compared to cleavage by RNase A, which are generated by 3'-P and 5'-HE, processing by RNase H, EDTA was added after post-translational incubation for destruction of ribosomes, and the reaction mixture was absoluely on column microcontroller and ADI high-10 (or microcontroller and ADI high-30). 2 μl of the mixture was combined with 18 μl of buffer RNase H (30 mm Tris-HCl, pH of 7.8, 30 mm (NH4)2SO4, 8 mm MgCl21.5 mm-mercaptoethanol and excess complementary DNA shunt), and the mixture incubated at 4C for 45 minutes. Then add the RNase H, and cleavage was carried out at 37C for 20 minutes.
Intramolecular or intermolecular mechanism of formation of the hybrid in the process of post-translational incubation
In addition to the above experiments, the authors present invention conducted a test to determine whether the reaction of formation of the hybrid, which runs at -20In the presence of Mg2+, intramolecular or intermolecular in nature. Free linker (dA27dCdCP or dA21C9C9With9dAdCdCP, where C9is O(CH2CH2O)3RHO2- jointly incubated with the matrix containing the DNA linker, but without puromycin at the 3'-end, in terms of translational and Petrakova any detected amount (i.e., less than 2% of the normal level)35S-Met, which gave grounds to assume that the post-translational formation of hybrid occurred mainly between the growing peptide chain and mRNA associated with the same ribosome.
In additional experiments, joint incubation was carried out with matrices and puromycin-oligonucleotides, hybrid and cross-linking products (matrix, hybridisable with incorrect protein) could be separated by electrophoresis. For all of the estimated matrices and linker combinations of any education crosslinking products were observed. In these experiments, hybrid crosslinking products can be formed through two different transoxania: (1) reaction of free matrices or linkers with peptide in complex peptide-mRNA-ribosome" or (2) reaction matrix, one complex with another peptide complex. One specific example of the analysis of the probability of the latter event is shown in Fig.24. Thus, the matrix lambda proteinopathy (-Ppaz), which synthesizes protein length 221 amino acid, were subjected to co-incubation with the ICC-matrix generating peptide of 33 amino acids. By themselves, both bladesa education only some hybrid products. After hybridization protein-proteinopathy with the ICC-matrix no education crosslinking products were observed. Similar experiments showed the absence of cross-linking products with some other combinations: ICC-matrix + matrix one codon; the standard linker + ICC-matrix in the ratio 20:1; and a flexible linker + ICC-matrix. These experiments are a powerful argument against the two possible TRANS-mechanisms of formation of the hybrid.
The influence of the length of the linker on the education of the hybrid was the same as in CIS-mechanism. Reducing the length of the linker from 19 to 13 nucleotides resulted in a sharp decrease in the expected number of hybrid product in the case when the length of the chain could no longer reach the peptidyl-transportnogo center from the decoding site (Fig.23). However, this effect can also be caused by occlusion of the puromycin inside the ribosome in that case, if the dominant is a TRANS-mechanism (for example, if the matrix associated with the ribosome, form the hybrid through a TRANS-mechanism). The reduction in the formation of a hybrid with longer linkers again is an argument against reactions of this type, because after the release of puromycin from the RIBO is tion
As demonstrated above, using a rolling linker and/or the exercise of the post-translational incubation in the presence of high concentrations MD2+the efficiency of formation of the hybrid grew by approximately 40% from the inclusion of mRNA. These results showed that one ml of the reaction mixture for broadcast in vitro can be generated up to 1014the hybrid molecules of mRNA-peptide, which gives the opportunity to produce pools of hybrid mRNA-peptide with very high multiplicity in order to use them in experiments in vitro selection.
Selective enrichment of a hybrid RNA-protein
The authors of the present invention have demonstrated the feasibility of using hybrids "RNA-peptide" in experiments on the selection and evolution by increasing the number of specific hybrid RNA-peptide from a complex pool of hybrids random sequences on the basis of the encoded peptide. In particular, the authors present invention was obtained the number of mixtures in which a small number of known sequence (in this case long ICC-matrix, LP154) was merged with a number of pool of random sequences (i.e., LP160). These mixtures were Translia the affinity of the oligonucleotide and the disulfide. Hybrids ICC-matrix was subjected to selective thus a monoclonal antibody against the ICC (Fig.16A). To determine the enrichment obtained in this selective stage, aliquots of the mixture hybrids "cDNA/mRNA-peptide before and after the thus amplified by PCR in the presence of radioactively labeled primer. Amplified DNA was digested with restriction endonucleases that cut the sequence ICC-matrix, but not the pool (Fig.16B and 16C). Determination of the quantitative relationship of the cut and uncut DNA showed that the ICC sequence was 20-40-fold enriched relative to the randomized library by thus.
These experiments were carried out as follows.
Reactions broadcast Reactions broadcast was carried out mainly as described above. In particular, the reaction was carried out at 30C for one hour in accordance with the manufacturer's recommendations (Novagen) and frozen overnight at -20C. Was obtained two versions of the six samples, one of which contained35S-methionine, and the other contained a cold methionine added to a final concentration of 52 μm. The reaction mixture 1-the moles matrix 25 µl reaction mixture.
Obtaining complex dt25-streptavidin-agarose
Complex streptavidin-agarose" (Pierce) was three times washed THOSE 8,2 (10 mm Tris-Cl, pH of 8.2, 1 mm EDTA) and resuspendable with getting 1:1 (vol./about.) suspension in THOSE of 8.2. Then 3'-biotinyl-T25synthesized using biomedi CPG (Glen Research) was added to the desired final concentration (usually 10 or 20 μm), and incubated with stirring for 1 hour. After that, the complex dt25-streptavidin-agarose three times washed THOSE of 8.2 and stored at 4With until further use.
Purification matrix of the translation reaction mixtures
To clean the matrices of the translation reaction mixtures were taken 25 μl of each reaction mixture was added to 7.5 ml of buffer allocation (1 M NaCl, 100 mm Tris-Cl, pH of 8.2, 10 mm EDTA, 0.1 mm DTT) and 125 μl of 20 μm dt25-streptavidin-agarose. The resulting solution was incubated at 4With under stirring by rotation for one hour. The tubes were centrifuged, and the eluent was removed. Then added 1 ml of buffer to allocate, suspension resuspendable, and the mixture was transferred into a 1.5 ml microcentrifuge tube. After that, the samples four times washed with 1 ml of the aliquot of ohla on the Millipore filter MC and suirable from dt25-agarose by washing with 2 volumes of 100 µl N2O, 0.1 mm DTT, and 2 volumes of 15 mm NaOH, 1 mm EDTA (4C) followed by neutralization.
This eluent was added 40 μl of a 50% suspension of washed dipropylacetamide (Pharmacia), and incubation was carried out at 4C with stirring for 1 hour. Then the samples three times washed with 1 ml THOSE of 8.2, and the eluent was removed. Then to solid substance (total volume approximately 20-30 ml) was added 1 ál of 1 M DTT, and the sample incubated for several hours, then removed and four times washed with 20 ml of N2(Total volume of 90 μl). Eluent contained 2.5 mm dipyrido, as it was shown by UV-absorption. 50 μl of this sample was precipitated with ethanol by adding 6 ál of 3 M NaOA, pH of 5.2, 10 mm spermine, 1 ál of glycogen (10 mg/ml, Boehringer Mannhiem) and 170 μl of 100% EtOH, and then incubated for 30 minutes at -70C and centrifuged for 30 minutes at 13000 rpm in microcentrifuge.
Reaction reverse transcriptase inhibitors
The reverse transcription reaction was carried out as precipitated by ethanol samples and on samples dipyridamole eluent as described below. For precipitated with ethanol samples of 30 μl resuspending matrix, N2O up to 48 ál and 200 picomol pscu was added to 16 μl of the buffer for the first circuit (250 mm Tris-Cl, pH 8.3, 375 mm KCl, 15 mm MgCl2supplied from Gibco BRL, Grand Island, NY), 8 μl of 100 mm DTT and 4 μl of 10 mm NTP and balanced when 42With, and then were added 4 μl of reverse transcriptase Superscript II (Gibco BRL, Grand Island, NY). To TR-separately eluent (35 ml) was added N2O (13 ml) and the reaction was carried out as described above. After incubation for one hour similarly numbered samples were combined (total volume of 160 μl). 10 μl of the sample was retained for PCR of each sample, not the last selection, and 150 μl of the sample was retained for thus.
To implement thus 170 μl of reverse transcription reaction Dvaleti to 1 ml of dilution buffer (10 mm Tris-Cl, pH of 8.2, 140 mm NaCl, 1% vol./about. Triton X-100) and 20 ál conjugate protein G/A (Calbiochem, La Jolla, CA) and pre-osvetleni by incubation at 4With rotation for 1 hour. Eluent was removed and added to 20 ál conjugate G/A and 20 μl of monoclonal antibody (1 μg, 12 picomol), then the sample is incubated with rotation for two hours at 4C. the Conjugate was inundated with microcentrifuge at 2500 rpm for 5 minutes, the eluent was removed, the conjugate was three times washed with 1 ml of aliquo 8,2, 100 mm NaCl. Related fragments were removed using 3 volumes of frozen 4% of the SPLA, and the samples were liofilizovane dry.
PCR samples and samples that have not undergone selection
PCR reactions were carried out by adding 20 μl of concentrated NH4OH to 10 µl of the coming of the material and only the selected material, and each mixture was incubated for 5 minutes at 55C, 70And 90For the destruction of any RNA present in the sample. Then, the samples were evaporated to dryness using a speed vacuum evaporator. 200 µl of the PCR mixture (1 μm primers 21.103 and 42.108, 200 μm dNTP in PCR buffer + MD2+(Boehringer Mannhiem) and 2 μl of Taq polymerase (Boehringer Mannhiem). 16 cycles of PCR were performed on sample No. 2, not the last selection, and 19 cycles were performed on all samples.
Then the samples are amplified in the presence of 5'-32P-labeled primer 21.103 in accordance with table 3 and twice purified separately using sets for direct purification of PCR Wizard (Promega) to remove all primers and shorter fragments.
32P-labeled DNA obtained for each of the above PCR reactions, on the basis of table 4. Total volume of each reaction was 25 µl. Each reaction mixture was added to 0.5 μl AlWnI (5 units (New England Biolabs)). Samples were incubated at 37C for 1 hour, and the enzyme was subjected to thermoinactivation by a 20 minute incubation at 65C. Then the samples were mixed with 10 μl of denaturing boot buffer (1 ml formamide ultra-high purity (USB), 20 ál of 0.5 M EDTA and 20 μl of 1 M NaOH) was heated to 90C for 1 minute, cooled and loaded on 12% denaturing polyacrylamide gel containing 8 M urea. After electrophoresis the gel was fixed with 10% (vol./about.) The SPLA, 10% (vol./about.) Meon, N2O.
Quantification of the cleavage
The number of ICC-DNA or DNA pool present in the sample, was estimated using the phosphor of the Visualizer (Molecular Dynamics). The amount of material present in each band was determined as the integral amount equal rectangles, built around the lanes of the gel. The total number of them./min registered in each band was calculated as the amount minus the background. Used three values of the background: (1) average for identical squares outside the square, kgs-band (band on the gel in this position is absent); and (3) the normalized value, which reproduce the closest match to 10-fold increments matrix between the coming tracks. Tracks 2, 3 and 4 in Fig.16B and 16C illustrate the enrichment of the target sequence compared to the sequence pool. Illustrated enrichment on track 3 (coming/selected) gave the highest value (17, 43 and 27 times more when using methods 1-3, respectively), due to the optimization of the signal-to - noise ratio for this sample. The obtained results are systematized in table 5.
In the second series of experiments, these same PUR-products once was purified using the kit for direct ocici PCR products Wizard, and the hydrolysates were quantitatively evaluated by the above method (2). In these experiments, similar results were obtained; for samples equivalent to the above samples in lanes 2, 3 and 4, it was determined to 10.7; 38 and 12-fold enrichment.
In vztro-selection from large libraries hybrids "RNA-peptide"
In another experiment, illustrating the selection of the right hybrid molecules from large libraries generated set of 21013randomized hybrids "RNA-the randomized codons, based on the scheme of the synthesis of 5'-(NNS)27-3' (where N represents an equimolar amount of A, G, C and T, a S is either G or C). Each NNS codon is a mixture of 32 triplets, which included the codons for all 20 natural amino acids. Conducting a randomized region was flanked by two binding sites with primers for reverse transcription and PCR, as well as sequences encoding the T7 promoter and the site of translation initiation. RNA synthesized by in vitr-transcription was modified by direct ligation of the matrix with oligonucleotide linker containing puromycin at its 3'-end, dA27dCdCP.
Purified legirovannye RNA was in vitro translated in the extract from rabbit reticulocytes with obtaining the hybrid RNA-protein" as follows: 123-dimensional DNA PP.01 (5'-AGC TTT TGG TGC TTG TGC ATC (SNN) 27 CTC CTC GCC CTT GCT CAC CAT - 3', N=A, G, C, T; S=C, G) (SEQ ID NO:34) was synthesized and purified on a 6% denaturing polyacrylamide gel. 1 nmol of purified DNA (61014molecules) amplified in 3 cycles of PCR (94C, 1 min; 65C, 1 minute; 72C, 2 minutes) using 1 μm of primers PIF (5'-AGC TTT TGG TGC TTG TGC ATC - 3') (SEQ ID NO:35) and RT (5'-TAA N 9,0, 0.1% Triton X-100, 2.5 mm MgCl2, 0.25 mm dNTP, 500 units of Taq polymerase, Promega). After precipitation of the DNA was again dissolved in 100 μl TE (10 mm Tris-HCl, pH to 7.6, 1 mm EDTA, pH 8.0). DNA (60 μl) was transcribed into RNA in the reaction (1 ml) using a kit for in vitro transcription Megashortscript from Ambion. The reaction mixture was extracted twice with a mixture of phenol/l3and excess NTP was removed by purification on column (NAP-25 (Pharmacia). Parametersetname linker 30-R (5'-dA27dCdCP) was synthesized as described in this application and attached to the 3'-end of the RNA library by direct ligation with the matrix. RNA (25 nmol) were incubated with equimolar amounts of the linker and shunt (5'-TTT TTT TTT TNA GCT TTT GGT GCT TG - 3') (SEQ ID NO:37) in the reaction mixture (1.5 ml) containing buffer for DNA T4 ligase (Promega) and 1200 units of DNA T4 ligase (Promega). After incubation at room temperature for 4 hours legirovannoi RNA was separated from religiouns RNA on 6% denaturing polyacrylamide gel, was suirable from the gel and again dissolved (200 μl ddH2O). To generate the hybrid molecules of mRNA-peptide legirovannye RNA (1.25 nmol) was translated in full 7.5 ml using a set containing rabbit reticulocytes, Rabbit Reticulocyte IVT from Ambion, in the presence of 3.7 µci35S-th concentration of 530 mm KCl and 150 mm MgCl2and incubated for 1 hour at room temperature. After completion of the reaction broadcast education hybrid amplified about 10 times the requested adding 530 mm KCl and 150 mm MgCl2.
Using this improved method was obtained approximately 1013purified hybrid molecules on one ml. Hybrids "RNA-protein was purified from the crude translation reaction mixture using affinity chromatography for oligonucleotides, and RNA portion of the attached molecules were subjected to reverse transcription, and then spent the selection stage using reverse transcriptase without RNase H as described below. Translated hybrid products were incubated with dT25-cellulose (Pharmacia) in buffer for incubation (100 mm Tris-Hcl, pH 8.0, 10 mm EDTA pH 8.0, 1 M NaCl and 0.25% Triton X-100, 1 hour at 4°C). The cellulose was isolated by filtration and washed with buffer for incubation and subsequent elution of hybrid products ddH2A. RNA was subjected to reverse transcription (25 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mm DTT and 0.5 mm dNTP, 2 units of reverse transcriptase Superscript II (Gibco BRL)) using 5-fold excess of "shunt" as a primer.
To study the effectiveness of technology is the major antibody against C-ICC using thus as a method of selection. Five repeated cycles of selection and amplification resulted in increased binding of the population of hybrid molecules with anti ICC monoclonal antibody E (Evan et al., Mol.Cell Biol. 5:3610 (1985)). In each of the first three cycles of selection was highlighted by elution of less than 1% of the libraries used for screening stage, however, in the fourth cycle of selection was allocated and polyinosine about 10% of the library that is associated with the antibody. In the fifth cycle of selection, the percentage of binding molecules was increased to 34%. This result is well consistent with the percentage with-ICC-hybrid constructs of wild type, which was associated with the antibody against C-ICC under these conditions (35%). In the sixth cycle of selection of any enrichment was not observed, and hybrid molecules obtained after the fifth and sixth cycles, used for characterization and determination of the sequence of selected peptides.
For these experiments the source library from 2 x 1013molecules were incubated with a 12-fold excess of s-ICC-binding antibodies E (Chemicon) in buffer selection (1x PBS, 0,1% BSA, 0.05% tween) for 1 hour atC. Complexes of peptide hybrid antibody" besieged by adding conjugate protein a - sepharose. After the addition is included in the stream (FT) was collected. Sepharose washed with five volumes of buffer selection (W1-W5) in order to remove non-specific binding substances, binding peptides were suirable four volumes of 15 mm acetic acid (E1-E4). cDNA-part buervenich hybrid molecules amplified by PCR and the resulting DNA was used to generate enriched populations of hybrid products, which were subjected to additional cycles of selection. To remove from the pool of peptides having affinity to protein a conjugate - sepharose, in the second round of selection was performed preliminary selection on the conjugate protein a - sepharose. The course selection was observed by determining the percentage of hybrid "35S-labeled RNA-peptide, which was suirable from immunoprecipitate acetic acid. The results obtained are shown in Fig.20.
A pool of selected peptides was demonstrated for specific binding of the antibodies against the ICC used for selection. Experiments on binding dehybridization peptides ongoing cycle 6 showed similar binding to the antibody, which was observed for the hybrid peptide, suggesting that part of the nucleic acid hybrid molecules is not necessary for a variety of conditions, thus, namely: (1) without antibodies against the ICC, (2) with a monoclonal antibody against integrin, ASC-3, which is the same isotype, but which does not bind to the epitope of the ICC, and (3) with antibody against myc, 9E10. The experiments were carried out by incubating35S-labeled hybrid products "RNA-peptide obtained in the sixth cycle of selection (0.2 pmol) in the buffer for selection (1PBS, 0,1% BSA, 0.05% tween) for 1 hour at 4With, or with the use of monoclonal antibodies against integrin4, ASC-3 (100 pmol; Chemicon), or without the use of this antibody. Complexes of peptide hybrid antibody precipitated with protein a conjugate - sepharose. After washing sepharose five volumes of buffer selection associated molecules were suirable the addition of 15 mm acetic acid.
In the control experiment without antibodies could not be detected any significant binding, which suggests that the selected peptides are not associated with nonspecific conjugate protein a - agarose. In addition, any binding with a monoclonal antibody against integrin were observed, which indicates that these selected peptides were specifically svi for to determine, communicate whether the selected peptide hybrid molecules with antigennegative site antibodies against myc, 9E10. If35S-labeled hybrid molecules obtained after conducting 6 cycles of selection were incubated with a monoclonal antibody against the ICC and with elevated amounts of unlabeled ICC-peptide, there was a decline in the percentage of binding molecules. These results are presented in Fig.21. This figure of 0.2 pmol35S-labeled hybrid products "RNA-peptide", the resulting six cycles of selection were incubated with 100 pmol of monoclonal antibodies against myc, 9E10, in the presence of 0, 0,2, 1,2, or 10 nmol of synthetic myc-peptide (Calbiochem). Complexes of peptide hybrid antibody" besieged by adding conjugate "protein a - sepharose". Values are presented as mean percentage of hybrid molecules that are associated with the antibody and which can be polyuretane 15 mm acetic acid, was determined in binding assays with three repetitions. Data competitive binding showed that the most dedicated hybrid molecules were specific to ICC-binding site.
Sequence analysis of 116 individual clones obtained were resolucoes twice and contained with-ICC-epitope of wild type, EQKLISEEDL (SEQ ID NO:2). The third sequence was almost identical to the other two, but found two point mutations in nucleotides, one of which was a mutation replacing Il for Val in a conservative region of the ICC-epitope. All sequences contained a "consensus" motif X (Q, EOXLISEXX (L,M) (SEQ ID NO:38), who had a great affinity with-ICC-epitope. The most highly conserved area was a "hard core" of four amino acids, LISE. In Fig.22 shows the amino acid sequence of the 12 selected peptides selected from a randomized 27-dimensional library. In the upper part of this figure shows the amino acid sequence with-ICC-epitope. Of these shows sequences included only sequences containing this consensus motif. Residues in the peptides that match the consensus, selected highlights. Clone R6-63 contained ICC-epitope of wild type. The remains of consensus (frequency >50% in this position) shown in the lower part of this figure.
Taking into account the fact that the conservative motif contained one amino acid that has been encoded to a specific 5'-primerno area, the authors of this application have determined that known with-ICC-epitope of 10 liblucene increased amount of wild-type epitope in five cycles of selection is clearly consistent with the enrichment factor >200 per cycle of selection, that is the factor, which was confirmed in a separate series of experiments.
Analyses for immunoprecipitation carried out on twelve selected sequences shown in Fig.22, confirmed the specific binding obtained from this library hybrids "RNA-peptide with antigennegative site monoclonal antibodies against the ICC. As hybrids "RNA-peptide", all twelve sequences were associated with the specified antibody against the ICC and did not show binding to the conjugate protein a - sepharose". Competitive binding to antibody against the ICC were also compared using35S-labeled hybrid products (derived from the twelve sequences) and unlabeled synthetic ICC-peptide. In used conditions labeled ICC-hybrid wild-type was contacted by 9% in the presence of unlabeled ICC-peptide, and the percentage of binding ranged from 0.4 to 12% for the tested twelve sequences. These data showed that these sequences are associated with the antibody against the ICC with affinato similar affinity ICC-hybrid wild-type.
Purification of peptides containing the sequence motif, and hybrids with immobilized RNA
RNA binding is t al., Science 273: 1547-1551 (1997)), containing the 3'-bitenova group, were synthesized using standartnoi chemistry using phosphoramidites. These synthesized RNA samples was unblocked, absoluely and subjected to gel purification as described in this application. Then 3'-biotinyl-RNA sites were immobilized by mixing the concentrated mother liquor RNA with 50% (vol./about.) suspension of streptavidin-agarose ImmunoPure (Pierce) in 1THOSE of 8.2 at the final concentration of RNA 5 mm within one hour (25(C) with shaking. Carried out two reactions broadcast containing (1) a matrix encoding a peptide fragment of 1 N, or (2) globin mRNA (Novagen) as a control. Aliquots (50 μl of 50% slurry.about.) each immobilized RNA was washed and resuspendable in 500 μl of buffer for binding (100 mm KCl, 1 mm MgCl2, 10 mm HepesKOH pH 7.5, 0.5 mm EDTA, 0.01% of NP-40, 1 mm DTT, 50 μg/ml yeast tRNA). Binding assays were carried out by adding 15 μl translation reaction mixture containing either N-peptide or globinemia matrix, in tubes containing one of the three immobilized binding sites, followed by incubation at room temperature for ml the buffer. Then add the RNase A (without Gnkazy, 1 μl, 1 mg/ml) (Boehringer Mannhiem) and incubated for one hour at 37For the release of related molecules. The supernatant was removed and mixed with 30 μl of the boot buffer with LTOs and analyzed by electrophoresis in SDS page with LTOstrizina. The same scheme is used to allocate the N-peptide hybrids, except that after the broadcast was added 35 mm gl2and then incubated at room temperature for one hour for stimulation of the formation of the hybrid.
The results of these experiments showed that the N-peptide retained its normal binding specificity as in the synthesis in vitro, and when it is generating in the form of a hybrid RNA hybrid with its own mRNA. This result was crucial. Attach a long nucleic acid sequence to the C-end of the peptide or protein (i.e. education hybrid) can lead to the violation of polypeptide function compared to dehybridization sequence. Due to its relatively high capacity for non-specific binding of nucleic acids, peptides enriched with arginine motif (ARM) give who what Intesa cDNA) retains the function of the free peptide, indicates that the specificity is maintained even in the case when there is a possibility of education or outspecifically or nonspecific complexes.
The use of systems of selection of proteins
The selection system of the present invention have commercial application in any field in which protein technology used in therapeutic, diagnostic or industrial problems. The technology selection is used for the improvement or modification of existing proteins, as well as to highlight new proteins with desired functions. These proteins may have a natural sequence, or they may have a modified shape, or they may be partially or fully synthesized sequence. In addition, these methods can also be used to highlight or identify valuable nucleic acids or small molecules to target.
The allocation of new binding reagents
In one specific embodiment, the technology of obtaining hybrids "RNA-protein", is described in this application can be used for separation of proteins with specific binding properties (e.g., binding with ligand). Proteins with the ability to Vysokoye is, which then allows use of the technology education of hybrid RNA-protein" instead of the traditional technology with the use of monoclonal antibodies. The reagents of the type of antibody, selected by this method can be used in any area where there are traditional antibodies, including diagnostic and therapeutic applications.
Improving human antibodies
The present invention can also be used to improve the human or humanized" antibodies for the treatment of diseases of any number. For this purpose were developed libraries of antibodies that were skanirovaniya in vitro, thus avoiding the necessity of applying techniques such as cell fusion or phage display. In one of its important applications of the present invention can be used to improve the libraries of single-chain antibodies (Ward et al., Nature 341:544 (1989); and Goulot et al., J. Mol. Biol. 213: 617 (1990)). For these purposes, the variable region can be constructed either from a human source (to minimize possible adverse immune reactions in the body), or it can contain a fully randomized cluster (to maximize the plurality of libraries). For screening in order to improve the molecules sovanny as shown in Fig.2). Then, as the level of selection progresses from stage to stage, for the next stage of binding using a higher degree of rigidity. To increase the rigidity changing conditions such as the number of stages of leaching, the concentration of excess competitor, conditions tebufelone, the duration of binding assays and selection matrix for immobilization.
Single-chain antibodies can be used either directly for therapy or indirectly for design of standard antibodies. Such antibodies can be used for various purposes, including the allocation of anticommunism antibodies, suppression of immune response and the development of vaccines against viral diseases such as AIDS.
The allocation of new catalysts
The present invention can also be used for selection of new catalytic proteins. In vitro selection and evolution were previously used for the selection of new catalytic RNA and DNA, as in the present invention they are used to identify new protein enzymes. In one specific example of this method, the catalyst can be selected directly by selection for binding to a chemical analogue of a phase transfer catalyst. In another specific example, CR, the use of the substrate associated with the affinity label) or by splitting (for example, by selecting for the ability to destroy specific communication and thereby to release the catalyst library members from the solid media).
This method of selection of new catalysts has at least two important advantages compared to the methods of catalytic antibodies (for review see Schultz et al., J. Chem. Engng. News 68: 26 (1990)). First, in a method of catalytic antibodies original pool are largely confined to typical immunoglobulin laying chain; in contrast, the original library hybrids "RNA-protein" can be either randomized or it may consist of, without limitation, variants known enzyme structures or protein cores. In addition, the selection of catalytic antibodies are usually based on the preliminary selection for binding analogs of the transition state with subsequent time-consuming screening for active antibodies; and again, in contrast, direct selection for catalysis can be carried out by a method using a library of hybrid RNA-protein", as was previously demonstrated using RNA libraries. In an alternative allocation method be the constituent selection.
Enzymes obtained by this method are highly valuable. For example, currently there is a critical need to develop new and effective industrial catalysts, which would accelerate chemical processes. The main advantage of the present invention is that it can be carried out in randomly selected conditions and is not limited to, for example, in vivo conditions. Therefore, the present invention facilitates the provision of new enzymes or improved versions of existing enzymes, which can have highly specific transformation (and thus minimizing the formation of undesirable side products), functioning in a pre-defined conditions, for example at elevated temperature, pressure or concentration of the solvent.
in vitro interactive trap
Technology using hybrids "RNA-protein" may also be used for screening cDNA libraries and cloning of new genes based interactions, "protein - protein". In this method, a cDNA library generated from the desired source (e.g., method Ausbel et al., see above, Chapter 5). For each cDNA candidate peptide acceptor (for example, tail puromycin) League is described above, then these hybrids (or improved versions of these hybrids are tested on their ability to interact with specific molecules, as described above. If necessary, this way you can avoid the use of stop-codons and 3'UTR regions either (i) the addition of tRNA suppressor for through reciting the stop area, or (ii) removing factor release from the reaction broadcast by thus, or (iii) combining (i) and (ii), or (iv) removing the stop codons and the 3'UTR of DNA sequences.
The fact that the stage of interaction occurs in vitro, allows careful control over the stiffness of the reaction using a non-specific competitor and related conditions, such as temperature and ion concentration. Modification of the normal small molecules using neytralizuet analogues (for example, the APR on gS) allows the selection that allows you to identify other conformers of the same molecule. This method can be used for both cloning and functional identification of many proteins as RNA sequence selected partner binding covalently linked, and therefore can be easily the man ENES, sequence which currently are determined in accordance with the "Project for the definition of the human genome".
The use of hybrids, "RNA-protein" format "microarray"
"DNA chips" consist of spatial deterministic matrices immobilized oligonucleotides or cloned fragments of cDNA or genomic DNA and used for rapid sequencing and profiling of transcripts. By annealing a mixture of hybrids "RNA-protein" (e.g., generated from the pool of cellular DNA or RNA) with such a DNA chip can generate a "protein chip display, in which each spot corresponding to one immobilized sequence capable of otjihase with the corresponding RNA sequence in a pool hybrids "RNA-protein". In this way the corresponding protein immobilized spatial certain way, depending on its relation with its own mRNA, and chips that contain a series of DNA sequences that show the corresponding series of proteins. Alternative peptide fragments of these proteins can be displayed, if the library proteins generated from smaller fragments of cDNA or genomic DNA.
Such an ordered display of proteins and peptides have metamodelo "protein - protein". In one particular form of this method, the protein-probe mark detectable label (e.g. a fluorescent dye), and the labeled protein is incubated with the protein chip display. In this way the identity of the proteins are able to bind with the protein-probe, determine the location of the spots on the chip, which becomes labeled by binding to the probe. Another application of this method is the rapid identification of proteins that have been chemically modified under the action of modifying enzymes (e.g., proteinkinase, acyltransferase and methyltransferases). By incubation of the protein chip display with the desired enzyme and radioactively labeled substrate with subsequent washing and autoradiography, can easily be determined localization, and hence the identity of these proteins that are substrates for modifying enzyme. In addition, the use of this method with the orderly arrangement of small peptides can further define the localization of areas such modification.
Technology protein display can be implemented using arrays of nucleic acids (including RNA, but preferably DNA), immobility is akih materials, as glass (for example, glass plates, silicon or silicon glass (e.g., microarrays), or gold (for example, gold plates). Methods for linking nucleic acids to specific areas on such hard surfaces, such as photolithographic techniques well known in the art and can be used for the production of solid media (such as DNA chips) for use in the present invention. Examples of methods that can be used for these purposes include, but are not limited to, the methods described by Schena et al., Science 270: 467-470 (1995); Kozal et al., Nature Medicine 2: 753-759 (1996); Cheng et al., Nucleic. Acids Research 24: 380-385 (1996); Lipshutz et al., BioTechniques 19: 442-447 (1995); Pease et al., Proc. Natl. Acad. Sci., USA, 91: 5022-5026 (1994); Fodor et al., Nature 364: 555-556 (1993); Pirrung et al., U.S. patent No. 6143854 and Fodor et al. WO 92/10092.
1. Method of producing libraries is iniziali broadcast and the start codon, functionally attached to the protein coding sequence and each of which is functionally attached to the peptide acceptor at the 3'-end of the protein coding sequence; and (b) in vitro translation protein coding sequences during incubation in the presence of monovalent cation concentration of at least 125 mm and/or bivalent or more high-valent cation concentration of at least 25 mm to obtain a population of hybrids "RNA-protein", thereby producing a library of proteins.
2. The method according to p. 1, wherein the monovalent cation is present at a concentration of approximately 125 mm - 1.5 M
3. The method according to p. 2, wherein the monovalent cation is present at a concentration of approximately 300 - 600 mm.
4. The method according to p. 1, wherein the monovalent cation is To+or NH+4.
5. The method according to p. 1, wherein the monovalent cation is PA+.
6. The method according to p. 1, characterized in that the incubation carried out at approximately room temperature.
7. The method according to p. 1, wherein the divalent cation is present at a concentration of about 25 to 200 mm.
8. The method according to p. 1, characterized in that collicello includes a stop sequence or DNA sequence or its analog, covalently linked to the 3'end of the RNA molecule.
10. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue has a length sufficient for the presence of the interval between the decoding site and the peptidyl-transferase center of the ribosome.
11. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately 60-70
12. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately less than 80 nucleotides.
13. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately less than 45 nucleotides.
14. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately 21-30 nucleotides.
15. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue is attached to the RNA molecule using a DNA-shunt".
16. The method according to p. 9, characterized in that the stop sequence or DNA sequence or its analogue contains a non-nucleotide composing the BUT(CH2CH2O)3RHO2(phosphate, poly (ethylene glycol).
18. The method according to p. 1, characterized in that the hybrid RNA-protein" also includes nucleic acid or its analog, located near the peptide acceptor, which increases flexibility.
19. Method of producing libraries of DNA that includes a stage (a) obtaining a population of RNA molecules, each of which includes a sequence of translation initiation and start-codon, functionally attached to the protein coding sequence and each of which is functionally attached to the peptide acceptor at the 3'-end of the protein coding sequence; (b) in vitro translation protein coding sequences during incubation in the presence of monovalent cation concentration of at least 125 mm and/or bivalent or more high-valent cation concentration of at least 25 mm to obtain a population of hybrids "RNA-protein"; and (c) generating a DNA molecule from each of the RNA portion of the hybrid with the production thereby of the library DNA.
20. The method according to p. 19, wherein the monovalent cation is present at a concentration of approximately 125 mm - 1.5 M
21. The method according to p. 20, characterized in that the monovalent cation prisutstviya To+or NH+4.
23. The method according to p. 19, wherein the monovalent cation is PA+.
24. The method according to p. 19, characterized in that the incubation carried out at approximately room temperature.
25. The method according to p. 19, wherein the divalent cation is present at a concentration of about 25 to 200 mm.
26. The method according to p. 19, wherein the divalent cation is MD+2.
27. The method according to p. 19, characterized in that each of the RNA molecule optionally includes a stop sequence or DNA sequence or its analog, covalently linked to the 3'end of the RNA molecule.
28. The method according to p. 27, characterized in that the stop sequence or DNA sequence or its analogue has a length sufficient for the presence of the interval between the decoding site and the peptidyl transferase center of the ribosome.
29. The method according to p. 27, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately 60-70.
30. The method according to p. 27, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately less than 80 nucleotides.
31. The method according to p. 27, characterized in that Leonidov.
32. The method according to p. 27, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately 21-30 nucleotides.
33. The method according to p. 27, characterized in that the stop sequence or DNA sequence or its analogue is attached to the RNA molecule using a DNA-shunt".
34. The method according to p. 27, characterized in that the stop sequence or DNA sequence or its analogue contains a non-nucleotide components.
35. The method according to p. 34, wherein the non-nucleotide component is one or more components BUT(CH2CH2O)3RHO2(phosphate, poly (ethylene glycol).
36. The method according to p. 19, characterized in that the hybrid RNA-protein" also includes nucleic acid or its analog, located near the peptide acceptor, which increases flexibility.
37. The method of selection of the desired protein from a library of proteins from p. 1 and a nucleic acid that encodes a desired protein, comprising the stages of (a) obtaining a population of RNA molecules of candidates, each of which includes a sequence of translation initiation and start-codon, functionally attached to the protein coding candidate sequence, and each of which is functionally recognize the Mering protein-candidate sequences at incubation in the presence of monovalent cation concentration of at least 125 mm and/or bivalent or more high-valent cation concentration of at least 25 mm to obtain a population of hybrids candidate RNA-protein"; and (C) selection of the desired hybrid RNA-protein, with the selection of the necessary protein and nucleic acid that encodes a desired protein.
38. The method according to p. 37, wherein the monovalent cation is present at a concentration of approximately 125 mm - 1.5 M
39. The method according to p. 38, wherein the monovalent cation is present at a concentration of approximately 300 - 600 mm.
40. The method according to p. 37, wherein the monovalent cation is To+or NH+4.
41. The method according to p. 37, wherein the monovalent cation is Na+.
42. The method according to p. 37, characterized in that the incubation carried out at approximately room temperature.
43. The method according to p. 37, wherein the divalent cation is present at a concentration of about 25 to 200 mm.
44. The method according to p. 37, wherein the divalent cation is Mg+2.
45. The method according to p. 37, characterized in that each of the RNA molecules of candidates further includes a stop sequence or DNA sequence or its analog, covalently linked to the 3'end of the RNA molecule.
46. The method according to p. 45, characterized in that the stop sequence or DNA sequence or its analogue has a length sufficient for the presence of the I, the stop-a sequence or DNA sequence or its analogue has a length of approximately 60-70
48. The method according to p. 45, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately less than 80 nucleotides.
49. The method according to p. 45, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately less than 45 nucleotides.
50. The method according to p. 45, characterized in that the stop sequence or DNA sequence or its analogue has a length of approximately 21-30 nucleotides.
51. The method according to p. 45, characterized in that the stop sequence or DNA sequence or its analogue is attached to the RNA molecule using a DNA-shunt".
52. The method according to p. 45, characterized in that the stop sequence or DNA sequence or its analogue contains a non-nucleotide components.
53. The method according to p. 45, wherein the non-nucleotide component is one or more components BUT(CH2CH2O)3RHO2(phosphate, poly (ethylene glycol).
54. The method according to p. 37, characterized in that the hybrid candidate RNA-protein" also includes nucleic acid or its analog,