Thrombin inhibiting dna-aptamers and method of structure stabilisation
SUBSTANCE: what is offered is a method of structure stabilisation of thrombin binding DNA-aptamers, and also DNA-aptamers stabilised in such a way. The presented method provides formation of an additional base-stacking system by means of heterocycles or their analogues by means of increasing a surface of an aromatic system of heterocycles or their analogues, owing to using methods of determining a tertiary structure or molecular simulation with stating the fact of contact formation of the aromatic system of heterocyclic bases or their analogues with a G-quadruplex quartet which is related to a lateral loop.
EFFECT: method allows more effective assembly of antithrombin DNA-aptamers and improved structural stability under physiological conditions.
7 cl, 7 dwg, 1 tbl, 2 ex
The invention relates to the field of biotechnology and medicine, and specifically to a method of stabilizing antithrombin DNA aptamers and new and stable antithrombin DNA aptamers with anticoagulant properties and antithrombotic activity, which can serve as a basis for the development of drug anticoagulant and antithrombotic drugs.
The level of technology
Thrombin is an enzyme of the class of hydrolases, most important component of the blood clotting system of human and animals. Blood is present in the form of an inactive precursor prothrombin and activated prothrombinase (active thromboplastin). According to the chemical nature of the thrombin - glycoprotein with molecular weight of about 40,000; contains about 5% carbohydrates. Thrombin belongs to the class of serine proteases (trypsin and others). Molecule of thrombin consists of two polypeptide chains connected by a disulfide bond: a-chain and B-chain, which is the active center of the enzyme and carbohydrate component. Thrombin exists in several active forms, which differ in the structure of the B-chain. The main function of the thrombin - conversion of fibrinogen to fibrin. Thrombin hydrolyzes four arginyl-glycine communication in the molecule of fibrinogen; hatshepsuts four peptide and is formed fibrin-monomer, which then aggregates the fibrin clot is, which is the basis of a blood clot. Reactions limited proteolysis involving thrombin is also accompanied by activation of factor XIII (fibrin-stabilizing factor) and factors V and VIII, participating in the reactions of the internal mechanism of blood clotting. With the participation of thrombin occurs platelet aggregation and compression (retraction) of a blood clot. It is shown that the relative abundance of thrombin in the body reflex activates the so-called anti-clotting system, while in the bloodstream received heparin and plasminogen activator, which are involved in maintaining the liquid state of the blood.
Thrombin is inactivated by diisopropylfluorophosphate, blocking the hydroxyl group of serine, which is included in the active center, and other inhibitors, are characteristic of a group of serine proteases. Blood thrombin is inactivated by antithrombin plasma: A2-macroglobulin, antithrombin III and / or heparin. Specific replacemeny thrombin inhibitor is a polypeptide hirudin contained in the salivary glands of the medicinal leech.
In recent years there has been developed a new type of inhibitors of thrombin - DNA-aptamers.
The aptamers are small molecules of nucleic acids, which can serve as high-affinity receptors for any of the targets from whole cells to low molecular weight organic compounds. Oligo is nucleotide aptamers with the desired properties isolated from libraries of random sequences by the methods of in vitro selection (SELEX), using their ability to interact specifically with the corresponding immobilized targets/ligands.
So, one of the first DNA aptamers inhibiting thrombin, was found Schultze R. et al. "Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG)", J. Mol. Biol. 1994 Feb 4; 235(5): 1532-47. DNA oligonucleotide dGGTTGGTGTGGTTGG, trombiculiasis an aptamer that specifically binds to thrombin and inhibits the activity of the enzyme in the chain of reactions that lead to the clotting of blood. The spatial structure of this terminzusage) can become the basis for the development of improved inhibitors of thrombin with similar structural motifs.
In the publication Nagatoishi (Nagatoishi S. et al., "Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions", Biochem Biophys Res Commun. 2007 Jan 19;352(3):812-7. Epub 2006 Nov 27) described antithrombine aptamers with G-QUADRUPLEX structure.
Gatto Century (Gatto C. et al., "Nucleic acid aptamers based on the G-quadruplex structure: therapeutic and diagnostic potential", Curr Med Chem. 2009;16(10): 1248-65) describes G-quadruplexes antithrombine aptamers, and also conducts a review of the current knowledge about the aptamers on the basis of G-QUADRUPLEX structures, as well as evaluating their diagnostic and therapeutic potential (as biotech drugs) for the detection and treatment of severe pathologies, including cardiovascular, cancer and viral diseases.
In addition, for the centre of the ina received attornye DNA biomedical properties of which are described, for example, in number of publications:
1. IE 920561 A1 (GILEAD SCIENCES) from 26.08.1992 year, which describes aptameric oligonucleotide that specifically binds with thrombin, and having a nucleotide sequence GGXTGG, where X is T, A, U, dU, or G, in particular, contains the nucleotide sequence GGTTGGTGTGGTTGG.
2. US 2005176940 A1 (UNISEARH LTD) from 11.09.2005 year, which describes aptameric oligonucleotide that specifically binds with thrombin, and having a nucleotide sequence GGTMGGXGGTTGG, where M is a or T, and X is a sequence of from 2 to 5 of any of the nucleotides or their modifications.
3. Griffin L.C. et al., "In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits", Blood. 1993 Jun 15; 81(12):3271-6, describes aptameric oligonucleotide having the sequence GGTTGGTGTGGTTGG.
However, the General lack intrinsic DNA aptamers, svyazyvaysya with thrombin, is their low stability in vivo.
IPCamera DNA dGGTTGGTGTGGTTGG (15TGT), specifically binds thrombin, was first obtained by the SELEX method (Bock L.C., L.C. Griffin, J.A. Latham, Vermaas EH, 'toole J.J.Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6; 355(6360):564-6). 15TGT - 15-tier thrombin - binding DNA aptamer was able to inhibit thrombin. Unlike widely used at the present time, thrombin inhibitors, such as Hepar is h, 15TGT has low immunogenicity and short lifetime in the body, it is a promising object to retrieve the drugs. 15TGT has a so-called intramolecular G-QUADRUPLEX structure formed by the two in the stacking G-quartets, one United TGT-loop and two TT loops.
The stacking interaction (by nature are van der Waals PI-e hydrophobic interactions) cause flat aromatic system of heterocyclic bases samouporyadochennoi in structures like stacks of the interplanar distance of 3.4-3.8 Angstrom. Thus there is a complete or partial overlap between the planes of the bases.
G-quartets that make up 15TGT consist of four guanine, United ecovalence using justinska hydrogen bonds. The Union of two G-quartets is called G-quadruplexes. G-QUADRUPLEX can be formed from two or four DNA molecules, giving intermolecular complexes. G-QUADRUPLEX have a characteristic range of circular dichroism.
Antithrombine aptamers 15TGT is the minimum monomolecular quadruplexes. In solution 15TGT loses structure QUADRUPLEX already at S, which is much lower than physiological temperatures.
It is known that potassium ions stabilize the structure of the G-QUADRUPLEX; complete with the Orc structure is implemented with high concentrations of potassium ions, while the physiological concentration is low and, as a rule, is only 5 mmol/liter (5 mm).
Thus, the technical problem of the present invention is to develop new ways of stabilizing the structure antithrombin DNA aptamers through the structure, and methods of design of stable structures of aptamers and their receipt.
The technical problem is solved by finding structural factors stabilize G-quadruplexes antithrombin DNA aptamers by molecular modeling and implementation in specific spatial structures. Based on the identified factors is the rational design of modified aptamers that are able to effectively communicate with thrombin. Is direct chemical synthesis of modified stable structures, compared to the efficiency of inhibition of thrombin compared with known aptamers.
The technical result of the claimed group of inventions is to improve the efficiency of Assembly antithrombin DNA aptamers and the stability of their structure under physiological conditions. The factors that determine positive effects for stabilization antithrombin DNA aptamers can be used for other G-QUADRUPLEX structures, including aptamers to other targets and for telomeric with whom rector.
The patent describes the development and construction of dynamic models of spatial patterns of DNA aptamers with the following primary structure:
- Single module amplified DNA aptamer (DTI-G84) NxGGGTTGGGTGTGGGNTGGGNy.
- Single module amplified DNA aptamer (DTI-A6) NxGTAGGTTGGTGNGGNTGGGGCNy.
- Two DNA aptamers with a modification of the loop (DTI-P91) NxGTAGGTTGGTGGGGNTGGRRCNx.
- Dvuhkanalnyy DNA aptamer (DTI-33) GGTTGGTGTGGNTGG-Tx-GGTTGGTGTGGNTGG.
- Four-module DEC - aptamers (DTT-r91) TNxGTAGGTTGGTGGGGNTGGRRCNxAGNyGTAGGTTGGTGGGGNTGGRRCNyC.
- Four-module DNA aptamer (DTI-Fx7) NxCTAGGTTGGATGGGNTGGTGNzGTAGGTTGGATGGGNTGGTCNy.
where N stands for any nucleotide selected from a, C, T, G; subscripts x, y,z denote any number of nucleotides that are not equal to each other; Txdenotes "x" consecutive residues; T, R denotes nucleotides purine base.
Appropriate oligodeoxyribonucleotides were synthesized by standard solid-phase synthesis. For all aptamers identified binding and inhibition of thrombin.
Brief description of drawings
Figure 1. The dependence of the thrombin time of the concentration of the aptamer in the plasma of man. 31TGT - aptamer comparison described in the literature (And novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm. Ikebukuro K., Okumura Y., Sumikura K., Karube I Nuleic Acids Res. 2005 Jul 7; 33(12):e108.), 15TCT - derived standard aptamer 15TGT, destabilized according to patent principles. DPI-P91 - aptamer, stable according to patent principles.
Figure 2. Schematic illustration of the spatial patterns described in the literature, the minimum G-QUADRUPLEX interacting with thrombin. Heterocyclic bases G1, G6, G10, G15 form the top Quartet of quadruplets. Heterocyclic base G8 is stacking with top Quartet.
Figure 3. A computer model of the spatial structure of the aptamer DPI-R stabilized according to patent principles. The Foundation of the G7 and G8 of the proposed structures and new G and A, highlighted in black, creating a large surface stacking interaction with the top Quartet.
Figure 4. The mobility of the atoms (fluctuations) in the spatial structure of the aptamers according to computational molecular dynamics of two antithrombine of aptamers. Gray shows data for patentable), and stabilized according to patent principles. Black color shows the data for the aptamer comparison found Ikebukuro and colleagues.
Figure 5. Schematic representation of the spatial structure of the proposed variants of the G-quadruplexes that interact with thrombin, and which are stabilized according to the patent principles. A. schematic representation of the aptamer DTI-A6. B) schematic representation of the aptamer DTI-G8. B) schematic representation of the aptamer DTI-33. G) schematic representation of the aptamer DTI-P91. D) schematic representation of the aptamer DTI-r9L (E) schematic representation of the aptamer DTI-Fx7.
According to preliminary x-ray analysis of the complex with thrombin aptamer from the aptamer in complex involved lateral loops TT, the conformation of which is determined by the structure of G-QUADRUPLEX. In this regard, any impact or modification, which results in stabilization of the structure of QUADRUPLEX affects the interaction of the loops with thrombin. The applicant has found that the increase in stacking interaction with the upper Quartet, causes the improvement of the thrombin-binding properties of aptamers due to the stabilization of QUADRUPLEX. Stabilization of QUADRUPLEX can occur for two reasons. First, by increasing the stacking interactions as such, including the effect of shielding from water heterocyclic bases of the top Quartet. Secondly, these stacking interaction limit the mobility of the ion, fixing and stabilizing it in the center of quadruplets.
First, let us consider the second reason. In addition to the described stacking interactions steric barriers to ion migration can be defined chemical groups sufficient the CSO size, correlated with the size of the Quartet 10 angstroms. Such blocking groups can be covalently or ecovalence associated with the upper hinge (T7-G8-T9 if 15TGT), closing the top Quartet (G1-G6-G10-G15). Alternative or additional arrangement of the blocking group may be covalent or non-covalent connection with the 5'- or 3'- ends of the oligonucleotide. Thus, the essence of the action of the blocking group is reduced to sterically prohibit migration of cations through the plane of the top quartets in the environment.
If you go back to the first reason stabilization QUADRUPLEX, it is determined by the efficiency of the stacking interactions with the top Quartet. In addition to blocking cation inside QUADRUPLEX it is possible to stabilize the structure by increasing the efficiency of the stacking interactions with the top Quartet. This can be done by adding nucleotides or other chemical groups capable of stacking-interactions with the 5'- or 3'-end, and chemical modification of the terminal nucleotide. All this leads to the formation of the hydrophobic core of the molecule aptamer and preventing access of water molecules as competitors hydrogen bonds nucleotides top Quartet. Isolation of hydrogen bonds quartets from access of water can increase the effective energy build up to 20 kcal/mol.
Detailed description of preferred the variants of implementation
In the following examples sets forth preferred materials and methods of the present invention.
Computer simulation of molecular dynamics (MD) allows to study in detail the factors affecting the stability of the structure or fluctuations of the atoms in the structure of biopolymers.
At the moment there are two variants of the static structures of aptamer TO data of nuclear magnetic resonance (NMR) and x-ray analysis (PCA). In the MD simulation of NMR model TV not undergone significant changes. In contrast, PCA-model TV changed its initial conformation - dimensional structure of G-QUADRUPLEX acquired hip geometry, unusual G-Quartet structures.
Despite the fact that the loops of the G-QUADRUPLEX structures are very difficult to model the MD with the help of modern computer power fields in NMR models of the Central loop TGT and side loops TT final dynamic patterns very similar to the patterns obtained on the basis of NMR spectroscopy. This positive result, you can go to build a dynamically stable models of different design choices 15TGT MD methods.
Option antiterminator of aptamers obtained To Ikebukuro et al., inhibited thrombin better than 15TGT, with its structure had additional nucleotides which both ends of the molecule. Methods of computer simulation model was constructed spatial patterns 31-tier) TGT: the ends of the molecules are locked into dutrieu structure and connected with quadruplexes module internal hinges on two nucleotides each (loop 2×2). For the obtained static model performed the MD simulation. Internal loop 2×2 can form additional interactions with the TGT loop with the participation of three nucleotides, which are located above the top Quartet. Characteristically, the nucleotide T9 upper loop is not involved in the formation of such characteristic patterns stacking interactions. According to patent the principle of stabilization of the structure due to the formation of strong stacking interactions this nucleotide T was replaced by the nucleotide G. guanine Residue forms a more stable stacking interaction and has more opportunities for the formation of hydrogen bonds. The resulting model is a new variant of the aptamer was subjected to MD simulations. The observation time (long path) amounted to 1 microsecond or 50 million iterations. In the observed time period model the spatial structure of a new variant of the aptamer showed increased stability in MD. Fluctuations of guanine residues belonging to quadruplets were significantly lower (Figure 4). The appearance of the second guanine in the Central loop allows the ILO to create additional interaction (Figure 3). All interactions were stable and did not change after reaching molecule equilibrium until the end of the trajectory. The plot hinges creates a structure in which all elements involved in the interaction. G-quartets are stable and are in parallel planes. Duplex plot also shows increased stability.
Determined the effectiveness of the inhibitory effect on thrombin new variant of the aptamer based structure which was based on patented principle. For this purpose, followed by chemical synthesis of a variant of the aptamer and its inhibitory ability in the hydrolysis of fibrinogen by thrombin.
Modified aptamer with a sequence of NxGTAGGTTGGTGGGGNTGGRRCNxwas assigned the index D11-P91.
Similarly, by means of computer simulation the MD method were obtained aptamers DTI-G84, DTI-33, DTI-r91, DTI-A6 and DTI-Fx7, the nucleotide sequence of which is disclosed earlier.
Synthesis of DNA aptamers according to the invention is carried out according to standard methods on solid media (Oligonucleotide Synthesis, M.J.Gait ed, 1984; Sambrook, Fntsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989).
The synthesis was carried out by solid-phase method on the automatic synthesizer, Applied BioSystems 380b using standard derivatives of nucleotides. As the polymer carrier used glass beads CG (control pore glass) with a pore size of 500 and 1000 Å. The first nucleoside C'-hydroxyl group was attached to the carboxyl group modified polymer carrier. Each synthesis cycle consisted of four stages:
- detritivore to remove dimethoxytrityl group treatment trichloroacetic acid and release of 5'-Oh groups;
accession nucleotide in the form of β-Tianeti-N,N-bis-diisopropylaminoethyl nucleoside;
- β-laitila group is removed after completion of the synthesis by treatment with ammonia. Antivirus reagent when condensation is tetrazole.
- caching. Join amidophosphate at the stage of condensation does not occur quantitatively, so about 2% of the 5'-Oh groups do not react. At subsequent stages of the synthesis of them can join the nucleotides that, ultimately, leads to gaps in the sequence of nucleotides. In this connection it is necessary to protect the free 5'-Oh groups, i.e. kierowanie, by reaction with acetic anhydride in the presence of N-methylimidazole.
oxidation Fofanova ether to phosphate iodine in pyridine.
To confirm the inhibition of thrombin-aptamers according to the invention and study of their anticoagulant activity was investigated inhibition of thrombin in seconds at a temperature of 270P in the system described in the table.
|Plasma human||2 ál|
|Thrombin human||13 IU|
|The aptamer||different concentrations|
Method spectrophotometric study of coagulation of human plasma in the presence of different concentrations of aptamer from 0 to 60 nm, the obtained values of inhibition of thrombin in seconds (see Figure 1).
Due to the fact that computer simulation is realistic options modified structures, but does not evaluate the effectiveness of the self-Assembly of polymer in the solution, the authors investigated the effectiveness of the claimed sequences as anticoagulant agents.
First option: Prothrombin time
Prothrombin time - time the formation of a clot of fibrin in plasma by adding thereto calcium chloride and tissue standard is sarounova thromboplastin. Prothrombin time characterizes the activity of the so-called prothrombin complex (factors V, VII, X and prothrombin factor II). The result is expressed in seconds (prothrombin time). Prothrombin test is a screening test that simulates the external path of blood clotting, so this test characterizes the first (protaminensulin) and second (combinatoria) phase plasma hemostasis and reflects the activity of prothrombin complex. The test is used to evaluate the shortage of factors of the prothrombin complex and external paths clotting. The results of the test, under normal content and quality of fibrinogen, dependant factors VII, V, X, II and their activity. The main factors of the prothrombin complex occurs in the liver, so this test is often used to assess its activity in the biosynthesis of protein.
More than standardized test is an international normalization ratio (MPE). In most cases when treated protivosvertyvayuschih drugs (anticoagulants) indirect action is sufficient to increase MPE in the range of 2 to 3, which corresponds to an increase in prothrombin time in 1,3-1,5 times in comparison with the initial value. Figure 6 shows the dependence of the prothrombin time from the concentrations of the different aptamer DNA is in human blood plasma.
Second option: Thrombin time
Thrombin test shows the time required for the formation of a clot of fibrin in plasma by adding thereto thrombin. This test characterizes the kinetics of the final stage of blood coagulation is the rate of conversion of fibrinogen to fibrin. Thrombin time depends only on the concentration of fibrinogen and activity of thrombin inhibitors (anti-thrombin III, heparin, paraproteins). Used for evaluation of the third phase of blood coagulation is the formation of fibrin, and condition of natural and pathological anticoagulants. Determination of the thrombin time is one of the most common methods of monitoring Eparistera and treatment fibrinolitikov, and is also used to diagnose hyperfibrinolytic States, afibrinogenemia and dysfibrinogenemia. Full incoagulability observed immediately after the intravenous administration of large doses of heparin and in the terminal phase of acute DIC. 7 shows the dependence of the thrombin time from the concentrations of the different aptamer DNA in the plasma of human blood.
Discussion of results
It is established that at low concentrations, a new variant of the aptamer DTI-R is a more effective inhibitor of thrombin in the reaction of hydrolysis of fibrinogen than the original aptamer 31TGT (Figure 1).
D is I justify the importance of the contribution of the stacking interaction of heterocyclic bases TGT loop from the top of the G-Quartet in the overall structure antiterminator Atamanova G-QUADRUPLEX comparison of the properties of aptamers, containing the replacement of the remainder of G With the TGT loop. The data obtained clearly demonstrate the importance of the stacking interaction. Direct replacement of the Central purine G in the loop TGT at least a heterocycle, pyrimidine, by definition, reduces the degree of stacking interaction, which leads to a considerable decrease in the degree of inhibition. Conversely, replacement of adjacent pyrimidine T TGT loop on the larger purine G leads to an increase in inhibitory activity of aptamer - increasing thrombin time. Similar to the effect of the stacking interaction can be modulated and derivatives of heterocyclic bases, and similar aromatic systems.
The importance of the described stacking interactions with the upper G-Quartet for the stabilization of the overall structure of the G-QUADRUPLEX indirectly confirmed by another fact. As previously noted, the traditional factor of stabilization of QUADRUPLEX is ion To+, which is coordinated by the oxygen atoms in the center of QUADRUPLEX and thereby stabilizes the overall structure. Coordination ion is dynamic in nature, i.e. ion mobile and can be exchanged with the external environment. The presence of additional stacking interactions are blocking the exit of the ion from QUADRUPLEX in solution through the top Quartet. The behavior of ion barium, BA2+differs from the behavior of the it To +the fact that his own coordination more effective and is stable in the structure of QUADRUPLEX. Additional stacking interaction between the upper Quartet, does not have a significant impact on the stabilization of the overall structure.
1. The method of stabilization of the structure of DNA aptamer, the main substructure which consists of QUADRUPLEX and two lateral loops TT, and which is able to specifically bind thrombin, including: the formation of an additional system stacking interactions with carbonyl compounds or their analogues by increasing the surface of the aromatic system of heterocyclic compounds or their analogues, due to the use of methods for the determination of the tertiary structure or molecular modeling with proof of education contact aromatic heterocyclic system substrates or their analogues with Quartet G - QUADRUPLEX, which is adjacent to the lateral loop.
2. Aptameric oligonucleotide DTI - G84 having antithrombotic activity, which contains a stable substructure obtained by the method according to claim 1, which represents oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
where N is any nucleotide from a number of G, a, T, C, and x and y are any number of nucleotides, to the which is not equal among themselves, and which specifically and vysokaye binds with thrombin.
3. Aptameric oligonucleotide DTI - R having antithrombotic activity, which contains a stable substructure obtained by the method according to claim 1, which represents oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
where N is any nucleotide from a number of G, a, T, C, x is any number of nucleotides, a R - denotes nucleotides purine base, and which specifically and vysokaye binds with thrombin.
4. Aptameric oligonucleotide DTI - A6 having antithrombotic activity, which contains a stable substructure obtained by the method according to claim 1, which represents oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
where N is any nucleotide from a number of G, a, T, C, and x and y are any number of nucleotides, which is not equal among themselves, and which specifically and vysokaye binds with thrombin.
5. Aptameric oligonucleotide DTI - r91, having antithrombotic activity, which contains a stable substructure obtained by the method according to claim 1, which represents oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
where N is any nucleotide from a number of G, a, T, C, x, and y is any number of nucleotides, which is not equal among themselves, a R - denotes nucleotides purine base, and which specifically and vysokaye binds with thrombin.
6. Aptameric oligonucleotide DTI - Fx7 has antimicrobial activity, which contains a stable substructure obtained by the method according to claim 1, which represents oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
where N is any nucleotide from a number of G, a, T, C, and x, y and z is any number of nucleotides, which is not equal among themselves, and which specifically and vysokaye binds with thrombin.
7. Aptameric oligonucleotide DTI - 33, having antithrombotic activity, which contains a stable substructure obtained by the method according to claim 1, which represents oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
where N is any nucleotide from a number of G, a, T, C, and x is any number of nucleotides, and which specifically and vysokaye binds to thrombin.
SUBSTANCE: method involves allele-specific Nested-PCR with primers which are matched with nucleotide sequences coding amino acids in positions 70-71 of the amino acid sequence. The allele-specific primers E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG - 3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC - 3' are matched with a part of BoLA-DRB3 gene located on chromosome 23 (localisation 23q21). They interact only with the nucleotide sequences coding alleles *11, *23, *28 =*7A causing genetic stability to cattle leukaemia. Then sequencing primer Zond 70/71 5'-GCCCGGCTACACCTGT - 3' is used to identify homo- or heterozygosity of an individual by the given alleles. If observing the primers interacting with alleles *11, *23, *28 =*7A, animals are considered to be leukaemia stable, while the absence of interaction with the same alleles can enable to refer to leukaemia unstable, and to neutral.
EFFECT: invention can be used for mass genetic typing of BoLA-DRB3 leukaemia tolerable animals in livestock and commodity economies for animal selection in a nuclear stock.
2 dwg, 4 tbl, 2 ex
SUBSTANCE: set contains species-specific oligonucleotide primer pairs and appropriate fluorescent-marked probes for conducting one-stage instant identification of several human-pathogenic Orthopoxviruses (VARV, MPXV, CPXV and VACV) by means of real-time multiplex PCR.
EFFECT: invention is intended for instant diagnostics of human and animal Orthopoxvirus infections by real-time multiplex PCR.
10 dwg, 2 tbl, 4 ex
SUBSTANCE: tissue is homogenised in a buffer and centrifuged at 105000 g for 60-90 min at 0-4°C to produce a cytoplasmic fraction which is then incubated with 10 mM of phosphocreatine and 10 mcg/ml of phosphocreatine kinase for 25-45 minutes at 35°C. Cytoplasmic fraction proteins are divided by ammonium sulphate at three stages, at the first stage ammonium sulphate is added to 38% of saturation and centrifuged to isolate a precipitate containing a 26S-proteasome pool, at the second stage, a supernatant is added with ammonium sulphate to 42 % of saturation and centrifuged to isolate a precipitate containing ballast proteins, at the third stage to the supernatant is added with ammonium sulphate to 70 % of saturation and centrifuged to isolate a precipitate containing a 20S-npoteasome pool. Ammonium sulphate is added in portions during 20 min on a magnetic stirrer and further mixed for 20 minutes.
EFFECT: invention allows dividing native 26S- and 20S-proteasomes and isolating them in those amounts they exist in living cells, with preserving at most an undamaged 26S-proteasome structure.
3 cl, 1 ex
SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.
EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.
87 cl, 37 dwg, 5 tbl, 6 ex
SUBSTANCE: method includes analysing aliquots of said sample by one or more methods of protein description specified in the chromatography. The method is based on genetic analysis techniques specified in RFLP and T-RFLP. These methods can be applied both separately, and in a combination. The offered methods allow obtaining the information on the presence and fractions of various individual proteins or coding sequences. The obtained information can be used for evaluating stability of a polyclonal cell line in process, and also estimating a structure of various parties of end polyclonal products.
EFFECT: methods allow describing the composition consisting more than of 10, 20 or greater number of antibodies.
16 cl, 18 dwg, 18 tbl, 14 ex
SUBSTANCE: composition includes at least three oligonucleotide probes and enables simultaneously determining a level of PSMB4, FCER2 and POU2F2 genes expression. The oligonucleotide composition under the invention is presented to be used, including as a part of a microchip, in a method for prediction of a developing disease in a subject suffering chronic lymphatic leukemia that involves analysing a level of expression of at least three named genes in patient's blood samples.
EFFECT: higher efficacy of the composition.
8 cl, 5 dwg, 16 tbl, 5 ex
SUBSTANCE: method provides recovery of DNA of an analysed strain followed by PCR with using nucleotide primers of terC, ilvN and inv genes of the following sequences: 89-S - AATCAAATCTCGCCCAGC, 89-As -GCTGCGTATCATTTCACC; 45-S - AGTGGTCTGCTTCTCTGG, 45-As -CGGCATACACAGAATACC; inv839 - TACCTGCACTCCCACAAC, inv1007 -CCCATACGCTGATCTACC. The analysed strains are differentiated by matching the sizes of the derived fragments of terC, ilvN and inv genes with the similar fragments in typical strains of principal and nonprincipal plague agents.
EFFECT: invention allows quick, effective and reliable differentiation of the strains.
1 tbl, 3 ex
SUBSTANCE: what is offered is a method of marking a human 7th chromosome providing in situ hybridisation of metaphasic or interphasic cell chromosomes of a tested sample and a DNA probe presented by a marker plasmid alpha R1-13 which consists of EcoR 1-EcoR l DNA fragment of a pBR 325 vector of the value 5966 base pairs and EcoRl-EcoRl alphoid DNA fragment of the human 7th chromosome of the value 680 base pairs. The testing environment in which the used DNA-probe specifically interacts with a centromeric region of the 7th chromosome without cross hybridisation with other human chromosomes are developed.
EFFECT: more efficient identification of the presented chromosome and enabled application of the new method in medical diagnostics.
SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.
EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.
6 cl, 1 dwg, 1 tbl, 1 ex
SUBSTANCE: during selection by markers, a set of alleles related to loci of quantity tokens (QTL) that contribute to expression of certain phenes of economic value is introduced into a corn idioplasm. The criteria are selected among grain crop capacity, grain moisture when picked, early and late root fit, stem drowning, frequency of common rust, frequency of ear rotting caused by Fusarium (ear wilt), resistance to Sulcotrione and panicle structure. Invention also relates to the method of such plants production, and also to methods of analysis and screening to identify plants with a required profile of alleles.
EFFECT: improved method of new corn plants production.
33 cl, 1 dwg, 19 tbl
SUBSTANCE: synthetic gene Ag85A Mycobacterium tuberculosis optimised for heterologous expression in nonpathogenic laboratory strains. The recombinant plasmid pAg85A-CBD consisting of an artificial bacterial operon of chimeric protein containing a promotor area of an early promotor of bacteriophage T5, chimeric protein gene and transcription terminator, a bacterial operon of beta-lactamase and a bacterial ColEl-type replication initiation site is produced. The invention also involves the Escherichia coli strain producing chimeric protein Ag85A-CBD, and also a method of immobilisation, concentration and cellulose purification of produced protein. Besides, the invention refers to the same recombinant protein Ag85A-CBD and an immunogenic composition containing it and used for tuberculous infection immunity induction.
EFFECT: invention allows preparing the producer strain providing a high production level of resistant immunogenic proteins which can be one-stage produced, immobilised and purified, producing the effective immunogenic compositions for tuberculosis.
7 cl, 2 dwg, 5 ex
SUBSTANCE: what is described is a diagnostic technique for deletions and duplications of PARK2 gene exons involving simultaneous amplification of one of park gene exons and a tracking gene in test samples of human genome DNA by real-time polymerase chain reaction with using exon-flanking outer primers and located between primers of fluorescent-marked hybridisation probes where polymerase chain reaction is four-staged in the following conditions: a reaction mixture is heated at temperature 50°C for 2 minutes, denatured at 95°C for 15 minutes, denatured at 95°C for 15 seconds, incubated at 61°C for 45 seconds; 35 cycles are repeated consistently with the two last stages; the absence of deletions, the presence of heterozygotic or homozygous deletions or the presence of duplications of exon is stated by the coefficient R which is calculated by formula R=2-(ΔCt), where ΔCt = [Ct tracking gene (reference DNA) - Ct park (reference DNA)] - [Ct tracking gene (patient's DNA sample)-Ct park PARK2 (patient's DNA sample)].
EFFECT: invention allows detecting mutations in PARK2 gene.
14 cl, 5 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine, and concerns oligonucleotide dosing. Substance of the invention involves application of at least one oligonucleotide of the sequence SEQ ID NO:5 for preparing a pharmaceutical preparation for prevention and/or treatment of a malignant tumour and/or metastases in a mammal, and said oligonucleotide is hybridised with a matrix RNA of TgF-beta2 gene and where said preparation contains said oligonucleotide in the concentration approximately 5 mcM to approximately 25 mcM.
EFFECT: lower preparation dose.
14 cl, 12 ex, 5 tbl, 5 dwg
SUBSTANCE: method of DNA-vaccine preparation modification is based on fusion of a non-structural protein NS1 TEV gene with a fragment of a mouse ornithine decarboxylase protein gene. The non-structural protein NS1 TEV gene is cloned in a plasmid used for protein expression in eukaryote cells. It is followed by site-directed mutagenesis of a 5'-terminal fragment of the viral gene for removing a terminating codon. The fragment of the ornithine decarboxylase gene coding 53 S-terminal amino acids and containing the terminating codon on the 5'-end is cloned. The plasmids are developed in E.coli cells and purified in the low endotoxin environment. It is followed by transfection by a plasmid preparation of eukaryote cell culture with immunoblot analysis of chimeric protein synthesis in cells. Mice are immunised to study the protective properties of the modified preparation.
EFFECT: invention allows higher effectiveness of the DNA-vaccine preparations, as the agents for prevention of development of tick-borne encephalitis.
5 dwg, 1 ex
SUBSTANCE: method provides transplantation of embryos all cells of which contain the additional genetic information to a recipient female. The introduction of the additional genetic information in a single-cell embryo is performed in a medium containing 150 mM NaCl, 2 mM HEPES and nucleic acid in the concentration 5 ng/mcl by generating two electric pulse trains of the filed intensity 100-200 V/cm of the duration 750 ms every 30 s.
EFFECT: method allows substantially increasing a number of produced transgene mice and reducing process length.
2 cl, 1 dwg, 2 ex
SUBSTANCE: recovered polynucleotide coding aminopeptidase containing a nucleotide sequence presented in the description is presented. Also, polynucleotide hybridised with said polynucleotide in the high stiffness environment is presented. An expression vector containing said polynucleotide, and an applicable host cell are described. Polypeptide related to the sequence presented in the description and being aminopeptidase is presented. A method of producing said polypeptide involving the stages of applicable host cell transformation by said polynucleotide or vector, cell cultivations in the medium adequate for said polynucleotide expression, and optional polypeptide purification from said cell or culture medium is offered. Besides, there has been described diagnostic technique for Aspergillus-infection in an organism involving the stages: a) recovery of a biological sample from said organism which is supposed to be Aspergillus infected, b) recovery of nucleic acid from said sample, c) determination whether said recovered nucleic acid contains polynucleotides hybridised with polynucleotide recovered from Aspergillus niger.
EFFECT: invention allows diagnosing Aspergillus-infection in the organism.
13 cl, 3 tbl, 11 ex
SUBSTANCE: invention concerns allele S608L (150C/T) of gene NOS2, and also application thereof as a diagnostic marker for evaluating the course of acute ischemic atherothrombotic stroke.
EFFECT: invention allows diagnosing the severity of the disease and thereby ensuring well-timed and adequate prescription of neuroprotective drugs selectively suppressing iNOS enzyme.
4 cl, 3 dwg, 2 tbl, 2 ex
SUBSTANCE: NA hybridisation is enabled by (a) heating of a hybridisation solution containing NA to temperature equal to or above than NA denaturation temperature that is followed with (b) cooling of the heated hybridisation solution to temperature whereat NA is hybridised with probes immobilised on a solid phase, and (c) hybridisation of NA contained in the cooled hybridisation solution with the probes immobilised on the solid phase at hybridisation temperature. According to the method, hybridised nucleic acids are transferred between denaturation, cooling and hybridisation zones; flow rate of the hybridisation solution with NA provides heating of the hybridisation solution at the stage (a) to temperature equal to or above than denaturation temperature with total heating time greater than denaturation time summerised with diffuse NA divergence time, and cooling at the stages (b) and (c) to hybridisation temperature with general cooling time at the stages (b) and (c) smaller than NA renaturation time.
EFFECT: method is completely compatible with a widespread microchip technology and enables hybridisation of both strands of two-strand DNA without preliminary run of one-strand DNA from two-strand one.
86 cl, 10 dwg, 2 ex
SUBSTANCE: biochip enabling diagnosing type I galactosemia is produced. Also, a method of using the produced biochip for detecting point mutations in GALT gene is offered. The given method involves a two-round multiplex PCR to produce a one-chained fluorescent marked DNA fragment; hybridisation on the biochip containing a set of oligonucleotides; analysis of the results.
EFFECT: invention extends the range of technological products used in diagnosing type I galactosemia.
3 cl, 34 dwg, 23 tbl, 6 ex
SUBSTANCE: recombinant plasmid pESAT6-CBD consisting of an artificial bacterial operon of chimeric protein containing a promotor area of an early promotor of bacteriophage T5, chimeric protein gene and transcription terminator, a bacterial operon of beta-lactamase and a bacterial ColEl-type replication initiation site is described. The invention also involves the Escherichia coli strain - producer of chimeric protein ESAT6-CBD, and also a method of immobilisation, concentration and cellulose purification of produced protein. Besides, the invention refers to the same recombinant protein ESAT6-CBD and an immunogenic composition containing it and used for tuberculous infection immunity induction.
EFFECT: invention allows producing the producer strain providing a high production level of resistant immunogenic proteins which can be one-stage produced, immobilised and purified, producing the effective immunogenic compositions for tuberculosis.
6 cl, 1 dwg, 5 ex
SUBSTANCE: invention relates to field of medicine and deals with optimised composition for RNA injection. Essence of invention includes application of mRNA and water injection buffer for preparation of solution for RNA injection, intended for transfer of mRNA and/or for its translation in host organism, injection solution includes Na+ with content 50 mM, Ca+ at least, 0.01 mM, content of K+ - 3 mM and optionally lactate.
EFFECT: increase of mRNA stability.
19 cl, 6 ex, 17 dwg