Method for determining modified nucleotides of rna

FIELD: biotechnology.

SUBSTANCE: selection of pair of oligonucleotide probes suitable for the study area of RNA, selection of a pair of donor and quencher of fluorescence with suitable optical properties is carried out. The direct chemical synthesis of modified probes is carried out, their effectiveness is assessed during melting with RNA containing known modifications. The obtained parameters of melting are assessed for each particular system. The control and the tested RNA are analysed, subjecting the mixture of probes and RNA to intense heating, slow cooling, and then the controlled heating with simultaneous detection of the fluorescence intensity, and the study parameters are selected according to the specific system. The results of melting are analysed, assessing the nature of the curves of melting, and the conclusion is made on the presence or absence of the modification in the test RNA in the presence or absence of modifications in the control RNA.

EFFECT: method is effective for detection of various types of RNA modifications and enables to detect the presence of modifications which do not prevent the formation of Watson-Crick pairs between nucleotides.

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The invention relates to the field of organic and medicinal chemistry, molecular biology and concerns a method for detection of modified bases in the composition of the RNA, which can be used for the rapid identification of RNA-modifying enzymes in the cell.

The natural nucleotides RNA often undergo various chemical transformations, and today we know more than hundreds of types of modified nucleotides (J. Rozenski, Grain P.F., J.A. McCloskey The RNA Modification Database: 1999 update. Nucleic Acids Res. 1999. 27(1): 196-7). Among all types of RNA of the most common modifications are methylation, pseudouridylation and digidropiridinovmi. It is known that some of these modifications provide chemical and functional diversity of RNA, improve their structural stability or functional activity (Bjork, G.R., et al. Transfer RNA modification. Anna Rev Biochem. 1987. 56:263-87; Dunin-Horkawicz, S., et al. MODOMICS: a database of RNA modification pathways. Nucleic Acids Res. 2006. 34 (Database issue): D145-9) and are even involved in the regulation of gene expression (Clancy MJ, Shambaugh ME, Timpte CS, Bokar J A. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res. 2002. 30(20): 4509-18).

Despite these data, there remains a vast range of modified nucleotides RNA function and the cause of which is still unclear. In addition, the implementation of the present invention POM who can find and peculiarities of regulation of the process of modifying the nucleotide RNA. For more than 50 years in the scientific world there are new methods that allow more or less successfully to determine the presence, quantity and even the position of one or other modifications in the RNA molecule. One of the first methods for the detection of methylated nucleotides was the analysis of the hydrolysis products of radioactively labeled RNA by two-dimensional electrophoresis (Sanger, F., G.G.Brownlee, and B.G.Barrell. A two-dimensional fractionation procedure for radioactive nucleotides. J Mol Biol. 1965. 13(2): 373-98). However, the method was extremely time-consuming, and the results were often ambiguous. As soon as Maxam and Gilbert proposed to analyze the DNA sequence by chemical and enzymatic hydrolysis (Mahat, A.M. and W.Gilbert. A new method for sequencing DNA. Proc Natl Acad Sci USA. 1977. 74(2): 560-4), appeared methods to identify some modified nucleotides RNA (Peattie, D.A. Direct chemical method for sequencing RNA. Proc Natl Acad Sci USA 1979. 76(4): 1760-4; Donis-Keller, H., A.M.Maxam, and W.Gilbert. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977. 4(8): 2527-38). These methods required working with radioactive isotopes, and analyzed the RNA molecule was not supposed to be long. In the early 80-ies of this method was adapted to long RNA molecules, suggesting to use oligodeoxyribonucleotide, complementary analyzed the sequences of RNA, followed by hydrolysis Rcason (Connaughton, J.F., et al. Primary structure of rabbit 18S ribosomalRNA determined by direct RNA sequence analysis. Nucleic Acids Res. 1984. 12(11): 4731-45). This allowed us to identify methylated at the 2'-hydroxyl groups of ribose nucleotides Cm, Am, Um, and nucleotides m⁷G. unfortunately, the improved method of hydrolysis with subsequent sequencing of RNA, was no less time consuming and has not been a universal finding any modifications of the RNA. In 1993 the group Operand has developed a new, still has not lost its relevance detection method modified nucleotides using the reverse transcription reaction (Bakin, A. and J.Ofengand. Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry. 1993. 32(37): 9754-62). It is applicable mainly to the presence of modified nucleotides that inhibit the formation of Watson-Cricova pairs (m₂⁶And, m³U, m¹G), and under special conditions the stopping reaction reverse transcription causes and the presence of a metal of the group on the 2'-hydroxyl of the ribose (Maden, V.E., et al. Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA. Biochimie. 1995. 77(1-2): 22-9). Using the unique chemical properties of some of the other nucleotides in combination with reverse transcription reaction can also detect pseudouridine Ψ (Bakin, A. and J.Ofengand. Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biocheistry. 1993. 32(37): 9754-62), m⁷G (Wintermeyer, W. and H.G.Zachau. A specific chemical chain scission of tRNA at 7-methylguanosine. FEBS Lett. 1970. 11(3): 160-164), m³C m²G (Mortimer, SA., J.S.Johnson, and K.M.Weeks. Quantitative analysis of RNA solvent accessibility by N-silylation of guanosine. Biochemistry. 2009. 48(10): 2109-14.), m⁵C (Rhodes, D. Accessible and inaccessible bases in yeast phenylalanine transfer RNA as studied by chemical modification. J Mol Biol. 1975. 94(3): 449-60; Negishi, K., et al A rapid cytosine-specific modification of E. coli tRNA Leu 1 by semicarbazide-bisulfite, a probe for polynucleotide conformations. Nucleic Acids Res. 1977. 4(7): 2283-92. Munzel, M., et al. Chemical discrimination between dC and 5MedC via hydroxylamine their adducts. Nucleic Acids Res. 2010. 38(21): e192; Clark, S.J., et al. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994. 22(15): 2990-7; Gu, W., et al. Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C. Mol Cell Biol. 2005. 25(18): 8191-201; Schaefer, M., et al. RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 2009. 37(2): e12.). It should be noted that 2'-O-methylated nucleotide is more stable under alkaline hydrolysis than demetilirovanny. This property is used in combination with reverse transcription reaction for the detection of nucleotide RNA containing metal group in the ribose (Maden, V.E. Mapping 2'-O-methyl groups in ribosomal RNA. Methods. 2001. 25(3): 374-82). Dihydrouridine also don't stop the reverse transcription reaction, so before further analysis using his unique ability to turn in the remainder of the β-ureidopropionic acid in alkaline medium (Xing F, Hiley SL, Hughes TR, Phizicky EM. The specificities of four yeast dihydrouridine synthases for cytoplasmic tRNAs. J Biol Chem. 2004.279:17850-60).

With the advent of new tools and approaches in the search for modified nucleotides RNA has changed. Thus, the method of mass spectrometry with electrospray-ionization (ESI-MS) provides a relatively mild conditions for the analysis of RNA (Kowalak, J.A., et al. And novel method for the determination of post-transcriptional modification in RNA by mass spectrometry. Nucleic Acids Res. 1993. 21(19): 4577-85), which in the form of multiply charged ions, and therefore, the maximum mass of the analyzed RNA significantly increased (Polo, L.M. and P.A.Limbach. Analysis of oligonucleotides by electrospray ionization mass spectrometry. Curr Protoc Nucleic Acid Chem. 2001. Chapter 10, Unit 10 2). With all the advantages of the method ESI-MS, it is often impossible to set the position of the modified nucleotide in the RNA molecule, and is almost impossible to position a modification inside of the nucleotide. In this regard, the method of mass spectrometry is used as a reliable complementary method to detect differences in the masses of fragments of RNA, not even reaching 1 Dalton.

Today widely used high-performance liquid chromatography (HPLC) to confirm the presence of a modified nucleotide composition of RNA. The separation of subject products are completely hydrolyzed RNA and the slightest changes in mobility there are various types of modified nucleotides. In addition, in combination with the method of the mass spectrum is metry exactly set the type and molecular weight of the investigated fragment (Kowalak, J.A., et al. A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry. Nucleic Acids Res. 1993. 21(19): 4577-85; Andersen, IE, B.T.Porse, and F.Kirpekar. A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA. RNA. 2004.10(6): 907-13; Giessing, A.M., et al. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria. RNA. 2009. 15(2): 327-36; Yan, F., et al. RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA. J Am Chem Soc. 2010. 132(11): 3953-64; Benitez-Paez, A., et al. YibK is the 2'-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA(Leu) isoacceptors. RNA. 2010.16(11): 2131-43; Havelund, JF, Giessing, A.M., Hansen, T., Rasmussen, A., Scott, L.G., Kirpekar, F. Identification of 5-hydroxycytidine at position 2501 concludes characterization of modified nucleotides in E. coli 23S rRNA. J Mol Biol. 2011. 411(3):529-36). However, to establish the exact position of the modified nucleotide using a combination of HPLC and ESI-MS is not possible. Not to mention sleek and sophisticated detection method 2'-O-methylated nucleotides, which is a combination of mass spectrometric analysis, the method of electron transfer (ET) and the dissociation activated by collisions (CAD), infrared multiphoton dissociation (IRMPD) or ultraviolet photodissociation (UVPD) (Smith, S.I. and J.S. Brodbelt. Hybrid activation methods for elucidating nucleic acid modifications. Anal Chem. 2011. 83(1): 303-10). Using this method are the different types of ions formed by the destruction of some preferred linkages in nucleic acids, the products of the gap in the application is to be placed IRMPD and UVPD differ and complement each other. The presence of 2'-O-methyl group prevents the formation of characteristic ribose types of ions, and the result of comparative analysis of mass spectrometry of the resulting RNA fragment establish the desired position of the methylated nucleotide in it.

Among all modified nucleosides greatest difficulty in identifying the cause dihydrouridine (D), monomethylarsonic on ekzoticheskom the nitrogen atom adenosine (m⁶A) and to a lesser extent, pseudouridine (Ψ). This is due to the fact that the formation of duplexes between the analyzed RNA and she complementary oligonucleotide they behave exactly the same as unmodified nucleosides. While D and Ψ have unique chemical properties and can be detected after chemical transformations by reverse transcription, then m⁶A until recently could detect only exhaustive hydrolysis with further analysis by means of HPLC and MS (Kawamura, Y. and Mizuno, Y. Studies on transfer RNAs. II. Modification of Escherichia coli formylmethionine transfer RNA. Biochim. Biophys. Acta. 1972. 277: 323-334; Limbach, PA., Grain, P.F. and McCloskey, J.A. Characterization of oligonucleotides and nucleic acids by mass spectrometry. Curr. Opin. Biotechnol. 1995. 6: 96-102). The problem is exacerbated because these three modified nucleoside most often found in the RNA of the cells of all living organisms. Was offered a rather interesting discovery method two modifica avannah nucleoside - Ψ and m⁶A (Dai, Q., Fong, R., Saikia, M., Stephenson, D., Yu, Y., Pan, T. and Piccirilli, J A. Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine. Nucleic Acids Research. 2007. 35(18): 6322-9). The principle of the method is the selection of a pair of complementary analyzed RNA oligonucleotides, one of which contains at the 5'-end of the so-called "binding residue", and shall also be radioactively labeled phosphorus atom in α-position. The second oligonucleotide hybridizes in the vicinity of "knowing". "Recognizing the balance is right across the analyzed nucleotide and takes different geometries, depending on the presence or absence of the modification. This affects the efficiency of ligation of two oligonucleotides, which are located opposite of the desired nucleotide, and thus indicates the presence or absence of modifications in RNA. The authors of this method for the first time showed how without additional chemical transformations can detect modified nucleotides Ψ and m⁶A. Shortcomings of the method is the use of radioactive isotopes, as well as non-quantitative yield of the reaction, which is markedly different for different sequences of the target RNA.

One of the methods of detection of modified nucleoside m⁶And used the group RAO, using antibodies specific to DNA containing monomethylamine the second ekzoticheskom the nitrogen atom adenosine (Banerjee, A., Rao, D.N. Functional analysis of an adaptive acid DNA adenine methyltransferase from Helicobacter pylori 26695. PLoS One. 2011. 6(2):e16810). Soon after, he was presented an improved method adapted for analysis of methylated RNA and called m⁶A-seq (Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M., Ungar, L., Osenberg, S., Cesarkas, K., Jacob-Hirsch j, Amariglio N, Kupiec, M., Sorek, R., Rechavi, G. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012. 485(7397): 201-6). The authors demonstrate that by using antibodies specific to the modified nucleoside m⁶A mRNA with subsequent exhaustive sequencing of its fragments, the opportunity to determine the entire set of desired modifications in all cellular RNA simultaneously. This method can be called a breakthrough, thanks to its versatility: it is possible to detect modified nucleotides, regardless of the sequence is RNA, it does not require additional processing RNA and work with radioactive isotopes. The only significant drawback of the proposed method is the need for expensive equipment, the acquisition of which can afford only a few laboratories in the world.

Closest to the claimed solution is the method of determining the methylation in the sequences of nucleic acids (Application on invention USA US 2010/0291565 Al). This image is eenie relates to a method of detecting DNA methylation preferably of natural origin by contact investigational nucleic acid with any chemical agent with the formation of the detected signal when their contact. The method includes a stage of denaturation of the studied DNA and detection obtained from chemical agent alarm at their contact. The level of methylation assessed by comparing the profiles of the curves of melting, in particular, by their inclination. In addition, the method also allows the use of advanced chemical agent in contact with the DNA and is able to extinguish the fluorescence arising from the application of the method.

The invention has significant differences from the known technical solutions. Namely, in the basis of the present invention is the hybridization of two oligonucleotides, one of which contains the donor fluorescence and the other cositel fluorescence, and has a large value of the ratio of the lengths of the oligonucleotides used. The proposed method is used to point modifications in certain nucleotides, in contrast to the prototype. The method aims to detect different types of modifications, including methylation, digidropiridinovmi and so on. Stage denaturation in the present study is preceded by a stage of hybridization of oligonucleotides, which increases the specificity of the detection method. The nature of the curves in the present invention may indicate different types of modifications and, thus, the possible determination of different types of modifications on the nome and the same nucleotide. In addition, the object of the study is RNA, not DNA.

All of the above differences profitable characterize the proposed invention, the application of which there is the possibility of detecting a wide range of modifications to specific nucleotides in the RNA molecule.

Thus, in this area remains a need to develop a universal method that would clearly, quickly and with minimal material costs to detect various modifications of the nucleotides in the structure of RNA.

The present invention is to develop a new simple and universal method for the detection of modified nucleotides in the structure of RNA.

The technical result of the claimed group of inventions is the ability to detect minimal differences in the structure of RNA, even for modifications not prevent the formation of Watson-Cricova pairs. In addition, the technical result consists in expanding the range of modifications that can be detected using the proposed method.

The problem is solved in that a method for detecting modified nucleotide in the composition of the RNA includes receiving control RNA; design of two oligonucleotide probes of different length, complementary to the analyzed RNA, with one of the probes having a smaller length, set mentoren RNA in the area, containing the analyzed nucleotide, and has a melting temperature less than the melting temperature of the second probe, one of the probes contains a molecule of the donor fluorescence and the other cositel fluorescence with the donor fluorescence and cositel matched with the provision of the overlapping wavelength range emitted by the donor fluorescence radiation with a wavelength range of radiation absorbed by tusitala fluorescence; mixing of probes with the test and control RNA, after which the mixture is subjected to heating to a temperature that provides a complete denaturation of the analyzed RNA and probes, slow cooling to the temperature optimum for the formation of stable duplexes between RNA and probes, and again slow and controlled heating with simultaneous irradiation and measurement of fluorescence intensity and obtain melting curves for the studied and the control RNA, and the conclusions about the presence or absence of modification in the tested nucleotide RNA are doing on the results of the comparison of the curves, and the coincidence of character curves, and/or the melting temperature make a conclusion about the presence / absence of modification in the studied RNA in the presence / absence of modification in the control RNA in the zone of hybridization of the shorter probe. As a control RNA using RNA with a known structure in IP is lleguemos region, and the analyzed RNA similar control or different from controlling the presence or absence of modifications in the test nucleotide. Study and control RNA is ribosomal or matrix or transport. The probes pick up from the condition of their hybridization to RNA in the immediate vicinity without overlapping. Longer probe pick with a melting point greater than the melting temperature of the second probe by more than 5°C. the First and second probes pick up the conditions of hybridization to the RNA at a distance from each other not more than 10 nucleotides between their immediate ends. The first and second probes pick up the terms of the arrangement of molecules of the donor fluorescence and tusitala fluorescence at a distance of not more than 10 nucleotides from each other by hybridization of probes to RNA. Molecules of the donor fluorescence and tusitala fluorescence to probe covalently attached. The probes consist of ribonucleotides and/or deoxyribonucleotides, and/or their analogs, and may include any modifications that do not affect the study. The mixture of probes with RNA carried out using a buffer solution. As the buffer solution may be used, comprising a substance that supports the solution pH in the range 6-8 .5, and salt of monovalent or multivalent, Katie the and. In one of the variants for mixing probes with RNA can be used 2-10 pmol RNA, 2-10 pmol of the oligonucleotide probe containing the donor fluorescence, 2-10 pmol probe containing cositel fluorescence, 2-10 μl of 5 × buffer for hybridization containing 200-300 mm Tris-HCl pH 8.3, 150 and 250 mm KCl and deionized water to bring the sample volume up to 10-50 μl. The mixture may optionally contain mineral oil in the amount of 3-10 mm. Heating and cooling of the mixtures is performed with a controlled speed, and the measurement of the intensity of fluorescence is carried out in the heating process. Possible use case, in which the initial heating is carried out until the temperature of the mixtures 70-95°C, slow cooling occurs at a rate of 0.5-4°/ min until a temperature of the mixture 30-10°C, re-heating of the mixtures produced with a speed of 0.5°/30 sec until the mixtures of 55-95°C. this cooling is carried out to a temperature which is below the melting temperature of the short probe is at least 5°C. reheating mixtures is carried out until the temperature, the value of which exceeds the melting point of the shorter probe, not less than 5°C. Obtained for analysis curves melting convert to curves derived fluorescence intensity on temperature temperature (differe the social curves).

Thus, the technical problem is solved by selecting a suitable for the study area RNA pairs of oligonucleotide probes, selection of a pair of donor and tusitala fluorescence with suitable optical properties. The length of the probes are selected experimentally, giving preference to those that provide the results of melting, interpreted in the best way. Is direct chemical synthesis of modified probes, the effectiveness is evaluated during melting with RNA containing a known modification. Estimated received the melting parameters for each specific system.

The invention is illustrated by drawings, where figure 1 presents the locations of the oligonucleotides containing the fluorophore and cositel fluorescence, hybridized to the RNA modification; figure 2 shows melting curves of RNA-DNA duplexes; figure 3 presents the differential melting curves of RNA-DNA duplexes.

The authors of the present invention have found that after hybridization in close proximity to each other investigated two complementary RNA oligonucleotide probes, one of which contains a fluorescent label and the other cositel fluorescence (Figure 1), thermodynamic parameters of this system can determine the presence or absence of modified bases in Uch is the site of RNA which hybridized shorter probe. Thermodynamic parameters were evaluated qualitatively in the slow heating of the entire system and a fixed detection level of fluorescence. At that moment, when one of the oligonucleotide probes begins to dissociate increases the distance between the donor fluorescence and tusitala, which leads to an abrupt increase in the intensity of fluorescence in the system. This jump occurs for each pair of probes and RNA at a specific temperature, which is considered the melting temperature of the duplex. In the experiment received the graphs of the level of fluorescence with temperature (melting curves), which were converted into curves derived fluorescence intensity on the temperature from the temperature differential curves). The area of the melting curves, in which the increase in fluorescence occurs abruptly (i.e. there is a point of inflection of the curve)correspond to peaks in the differential curve.

According to the claimed invention oligonucleotide probe, complementary to the analyzed RNA in the immediate area containing the analyzed nucleotide, should have a melting temperature below the melting temperature of the second probe. It does not matter which of the probes modified by the donor, and which is tusitala is luorescence. When comparing the parameters of the system containing modified and unmodified RNA, in most cases, there is a difference in the melting temperature and the nature of the melting curves. Moreover, differences in melting temperature can be less than 1 degree. This is due to the characteristics of each individual system, which, depending on the presence or absence of modifications can be stabilized or destabilized by changes in the geometry of the duplex and the nature of the relationship between probes and RNA in the area of the analyzed nucleotide. The probes can be a modified oligoribonucleotide, oligodeoxyribonucleotide may also contain any modifications that do not affect the ability of the probes to the hybridization.

The authors have obtained melting curves for systems with ribosomal RNA, which contained the following modified nucleosides: pseudouridine Ψ, dihydrouridine D, methylated 6-position of adenosine m⁶And, methylated at the 7 position guanosin m⁷G, methylated at the 2'-O-hydroxyl of the ribose nucleosides Bm. Of these modified nucleosides using the proposed method were detected D, m⁶A and m⁷G, the difference in melting temperature with non-modified RNA was 4 degrees, with the nature of distorting the x melting during detection m ⁶And was significantly changed, which simplified the analysis. It should be noted that modified nucleosides Ψ and Bm did not cause a noticeable shift of the melting curves. In all likelihood, pseudouridine slightly changes its environment compared to uridine, and thermodynamic parameters duplexes formed by them are exactly the same. Methylated ribose nucleosides Tue, though contain additional hydrophobic group, which undoubtedly affects the structure of the DNA-RNA duplex, however, do not show any peculiarities in the course of melting of the proposed method.

Also unique is the principle of selection of oligonucleotide probes for the implementation of the method, which is complementary RNA in the area of the proposed modified nucleotide, and one of them contains cositel fluorescence, and the other donor fluorescence.

The best result is achieved by observing the following conditions:

for detection of modified nucleotides need exactly two oligonucleotide probe;

one of the used probe should be complementary to the region of the investigated RNA so that in this area was the target nucleotide;

- probe, complementary to a region of RNA containing the target nucleotide, must have a melting point significantly lower than the temperature has been melted down the I of the second probe (at least 5°C);

- the second of the used probe should be complementary to the analyzed RNA in close proximity to the first probe so that there was no overlapping probes, and their immediate ends located on RNA not later than 10 nucleotides;

one of the probes should be modified molecule of the donor fluorescence and the other probe should be modified molecule of tusitala fluorescence with hybridization probes for RNA molecules of the donor and tusitala fluorescence should be placed at a distance of not more than 10 nucleotides;

- pair donor and tusitala fluorescence used in the present invention must be compatible, i.e. the wavelength range emitted by the donor radiation must at least partially overlap with the wavelength range absorbed by tusitala;

the probes can comprise ribonucleotides or deoxyribonucleotides;

the probes can contain any modifications which do not prevent the carrying out detection.

Below is a more detailed description of the proposed method.

The probes according to the invention can have a length of from 8 to 35 nucleotides, and a probe complementary to the RNA in the area of the analyzed nucleotide, should have a melting point at least 5°C below the melting temperature of the second probe. One of the probes must contain covalently SV is related to him the donor molecule fluorescence, and the other probe of the pair must contain covalently associated molecule tusitala fluorescence. In some embodiments, the implementation of the probes may contain additional, complementary to the sequence of RNA nucleotides or derivatives thereof, and may contain modified mezhnukleotidnyh connection.

Below are used in the invention terminology.

The term "RNA" understand ribonucleic acid. In the framework of the present invention the object of the study is any RNA of natural origin.

The term "DNA see deoxyribonucleic acid. Oligonucleotide probes are often the DNA.

The term "modified nucleotide" means a nucleotide derived from one of the nucleotides, the nitrogenous base is uracil, thymine, adenine, cytosine or guanine, which differ from the presence or absence of Vice or nature of the formed links.

The term "strain wild-type" refers to the phenotype inherent in the majority of individuals in natural populations of this species.

The term "RNA wild-type" refers to RNA isolated from wild-type strain.

The term "mutant RNA" refers to RNA that is different from the RNA of the wild

type.

The term "total RNA" under asomewhat set of different RNA isolated from cells at the same time.

The term "ribosomal RNA" means ribonucleic acid of natural origin, which is part of the ribosome.

The term "messenger RNA" means ribonucleic acid of natural origin containing information about the primary structure (amino acid sequence) of proteins.

The term "transfer RNA" means ribonucleic acid of natural origin, which function is to transport amino acids to the site of protein synthesis.

The term "oligonucleotide probes" mean oligoribonucleotide or oligodeoxyribonucleotide that are specifically associated with a specific sequence of the target RNA or DNA. Oligonucleotide probes often contain various modifications, for example, molecules of the donor fluorescence, the phosphoric acid residue with isotope³²P, Biotin and others, depending on the future goals of the experiment. Appropriate oligodeoxyribonucleotide were obtained using standard solid-phase synthesis.

The term "duplex" understand the structure of the two chains of nucleic acids, forming a double-stranded helix, held mainly canonical Watson-World interactions.

The term "hybridization" understand the process of selective binding to the RNA and oligonucleotide probe according to the principle of complementarity. In experimental conditions, the hybridization occurs most effectively when strong heating up to 80°C, followed by slow cooling to room temperature.

In the framework of the present invention, the term "fusion" refers to the process of heating hybridized nucleic acids to the complete dissociation with the formation of the individual molecules in solution. The melting of nucleic acids, and other polymers, may be stepped, with the formation of intermediate stable complexes due to the rearrangement of nucleotide residues and the formation of new stabilizing relations, for example hydrogen.

The term "melting temperature" means the temperature at which the structure of the cooperative is transferred in the molten state. The duplexes formed nucleic acids, there may be several such points cooperative transition. In the framework of the present invention during melting patterns observed a sharp flare-up of fluorescence, which indicates the simultaneous transition of the duplex formed by the shorter oligonucleotide, in the molten state.

Under "RNA sequence" in the present description polymer understand the sequence of ribonucleotides and their derivatives. Nucleotide - sugar to the 5'-carbon atom of which Pris the United phosphate group, and to the 1' carbon atom attached to the nitrogen base. Sugar is ribose or deoxyribose (in DNA and RNA, respectively). Grounds can be adenine, guanine, thymine, uracil and cytosine, and their derivatives. In the sequence of the probes can be included aromatic group, non-specified reasons.

The term "nucleotide" in the present description understand nucleotides, which can be natural nucleotides (such as ATP, TTP, GTP, P, UTP) or modified nucleotides. Modified nucleotides are nucleotides containing bases, such as, for example, adenine, guanine, cytosine, thymine and uracil, xanthine, inosine, and cousin, which has been modified by substitution or addition of one or more atoms or groups. Some examples of modifications that may contain nucleotides, the basic groups are modified include, but are not limited to, alkylated, halogenated, etiolirovannye, aminirovanie, amidarone or acetylated Foundation, in various combinations. More specific examples include 5-propenylidene, 5-propenylidene, 6-methyladenine, 6-methylguanine, N6,N6-dimethylamine, 2-propylidene, 2-propellane, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having modify the paths in the 5-position, 5-(2-amino)propylurea, 5-halogenation, 5-halogenides, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethanol, 5-methoxyuridine, deadenylation, such as 7-deaza-adenosine, 6-azauridine, 6-azacytidine, 6-Osotimehin, 5-methyl-2-thiouridine, other thio-base, such as 2-thiouridine and 4 thiouridine and 2-thiocytidine and dihydrouridine, pseudouridine, queuosine, naphthyl and substituted nattylove group, any O - and N-alkylating purines and pyrimidines, such as N6-methyladenosine, 5-methylcarbamoylmethyl, uridine 5-oxiana acid, pyridine-4-one, pyridin-2-it, phenyl and modified phenyl group, such as aminophenol, or 2, 4, 6-trimethoxybenzoyl, 8-substituted adenine and guanine, 5-substituted orally and timiny, isoperimetry, carboxyhydroxymethyl nucleotides, carboxymethylaminomethyl nucleotides and alkylarylsulfonate nucleotides. Modified nucleotides include nucleotides that are modified by a sugar group (for example, 2'-fluoro or 2'-O-methyl nucleotides), and a nucleotide having a sugar, or their equivalents, which are not ribosom. For example, the sugar group may be, or may be based on mannose, arabinose, glucopyranose, galactopyranose, 4'-chiriboga and other sugars, GE is erotico or carbocyclic. The term "nucleotide" also means a universal basis. As an example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole or nebularis. Modified nucleotides include labeled nucleotides, such as nucleotides labeled with a radioactive label, a fluorescent label, a molecule tusitala fluorescence, enzyme or pigment.

Under the "modified mezhnukleotidnyh links see all modified mezhnukleotidnyh connection known in this field and who are specialist in this field will find it possible to use in the context of the present invention. Modification mezhnukleotidnyh ties include, but are not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, 5'-alkylenediamine, 5'-methylphosphonate, 3'-alkylenediamine, avirati boron TRIFLUORIDE, esters of boron and phosphorous acid and selenophosphate 3'-5' link or 2'-5' communication, phosphocreatine, cyanoacetylurea, hydroporinae communication alkylphosphonate, alkylphosphonate, arylphosphonate, phosphorothioate, phosphorodithioate, phosphinate, phosphoramidate, 3'-alkylphosphonate, aminoalkylphosphonic, conopophagidae, phosphorodiamidate, phosphorotrithioate, phosphoroamidite, ketones, sulfones, sulfonamides, carbonates, carbamates meilinger, is etilendiamintetra, formacelli, thioformate, oximes, methylaniline, methylenediamine, thioimidate, communication with rebatedelivery groups, aminoethyl glycine, Silovye or siloxane bond, alkyl or cycloalkyl connection with or without heteroatoms, for example, from 1 to 10 carbons which may be substituted or unsubstituted and/or substituted and/or contain heteroatoms, morpholinomethyl, amides, polyamides, where the base can be attached to the Aza nitrogen of the main chain directly or not directly, and combinations of such modified mezhnukleotidnyh ties.

In the invention according to the claimed solution, the probes may be associated with conjugate, giving them additional properties. The conjugates may include, but not be limited to, for example, amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, hydrocarbons, polymers, such as polyethylene glycol and polypropylenglycol, as well as analogs or derivatives of all these classes of substances, cholesterol, phospholipids, di - and triacylglycerol, fatty acids, hydrocarbons, which are optionally contain substituents, enzymes, Biotin, digoxigenin and polysaccharides.

Probes must contain covalently attached molecule fluorophore or tusitala. Used fluorophores may include, but are they not the OTF is to socialisa: FAM, ROX, Alexa Fluor, TAMRA, BODIPY derivatives tsianina, such as SS3 or So Dabsyl, or other suitable fluorophores are known in the prior art. Used extinguishers may include, but not be limited to: all derivatives BHQ, Qxl, Iowa black FQ, Iowa black RQ, IRDye QC-1. Such conjugates can be attached to either end of the oligonucleotide, and also to any intermediate nucleotide, if the distance between the two conjugates liberalizovannykh probes does not exceed 10 nucleotides.

To implement this method, the probes can be synthesized by any method known in this field. In one of the embodiments, the probes can be obtained by chemical synthesis of oligonucleotides and/or legirovaniem short oligonucleotides.

The invention can be used to detect RNA owned by some pathogens that are resistant, for example, to macrolide antibiotics. Such methods may be used in a clinical setting to detect RNA of pathogens modifications, providing resistance to antibiotics, in particular, to detect demetilirovania nucleotide A 23S ribosomal RNA of the bacteria, leading to their resistance to antibiotics of macrolide number, such as erythromycin.

The invention is illustrated specific implementation options that are not intended to limit the claimed technical the CSO solutions.

Step 1. Synthesis oligodeoxyribonucleotide probes.

Synthesis oligodeoxyribonucleotide probes 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).

Probes containing at the 5'end of the fluorophore FAM, were obtained by the method of automatic solid-phase synthesis synthesizer MerMade 48 using as reagents amidophosphate nucleosides. As a carrier was used, the polymer is based on SiO₂with controlled pore size (CPG - Controlled Pore Glass, GlenResearch). The column with the carrier contained the first nucleotide of the probe that is attached to aminopropionic group on the surface of the media using succinylcoa linker linking the amino group and the 3'-hydroxyl group of the nucleotide. On each cycle of the reaction was detritivorous product, then spent the build-up circuit activated amidophosphate. Unreacted 5'-hydroxyl group of the growing chain blocked with a mixture of acetic anhydride and 1-methylimidazole, and linked to the substrate, the product was treated with an aqueous solution of iodine and pyridine. At the final stage with the obtained oligonucleotide was removed protective group DMT 2% trichloroacetic acid in dichloromethane and modified received oligonucleotide by amedica 6-FAM. Oligonucleotid the s was removed from the polymeric substrate with a saturated solution of ammonia at 55°C for 3 hours, and remove the protective group.

For solid-phase synthesis of probes with one molecule of tusitala on the 3'-end used a standard polymer 3'-BHQ-1 CPG (glass with controlled pore size and modification BHQ1, GlenResearch). Thus, at the first cycle of synthesis product already contained a molecule of tusitala BHQ1. Synthesis of oligonucleotides containing cositel, made standard by the method described above.

The resulting oligonucleotides were purified by electrophoretic separation of the reaction products in polyacrylamide gel.

Step 2. Isolation and purification of RNA.

To obtain the total RNA according to the invention, suitable for analysis by the method of melting was performed the following procedures:

Cells of the investigated strains were sown with wastewater stroke before the formation of isolated colonies on agar medium, incubated overnight at 37°C, then a separate colony was transferred into 1 ml LB overnight culture and again incubated with vigorous shaking overnight at 37°C.

30 µl of the overnight culture was inoculable in 30 ml of LB. The cells gave rise to achieve the optical density And₆₀₀=0.6 and was cooled in ice at 4°C for 20 minutes the Cells were collected by centrifugation at 6000 rpm in a JA10 rotor for 10 min, washed with buffer A (20 mm Hepes-KOH pH 7.5, 6 mm Mg(OAc)₂, 150 mm NH₄Cl, 4 mm β-mercaptoethanol, 0,05 mm spermine, 2 mm sperme is in) and centrifuged at 6000 Rev/min

The obtained wet cells can be frozen in liquid nitrogen and stored at T=-80°C.

Precipitated cells resuspendable in 1 ml of cold buffer a and was destroyed by ultrasound (6 times for 30 sec). Debris was separated by centrifugation at 15,000 rpm for 40 min in the rotor JA-20. The supernatant decantation, and thereto was added an equal volume of buffer for extraction In (300 mM NaOAc, 25 mm H₃BO₃pH 7.0, 0.5 mM SDS, 5 mm EDTA). To the solution was added 1 volume of phenol, which was intensively for at least 30 sec, centrifuged in a tabletop centrifuge at 13,000 rpm for 10 minutes. The upper, aqueous phase was collected and again was extracted with 1 volume of phenol, and then 1 by volume mixture of phenol and chloroform in the ratio of 1:1. To the aqueous phase was added to 3 volumes of ethanol and precipitated at -20°C for one hour. Precipitation was centrifuged in a tabletop centrifuge at 13,000 rpm for 10 minutes and then dried in a vacuum evaporator or air. The obtained precipitation total RNA was dissolved in a mixture of 50 μl of deionized water and 50 μl of buffer, and then was extracted with a mixture of phenol and again besieged RNA three volumes of ethanol at -20°C for 1 hour. Precipitation was dissolved in 50 μl of deionized water and measured the concentration of rRNA spectrophotometrically at a wavelength of 260 nm.

The concentration of total rRNA was calculated on the basis of its share in the total mixture is th cellular RNA (which is 0.8), according to the formula:

OD₂₆₀[PU/ml]∗0,0258[nmol/th]∗0,8=(rRNA)[pmol/ál]

The obtained solutions total rRNA in the aliquot of 10 μl were frozen in liquid nitrogen and kept at a temperature of -80°C.

Step 3. Obtaining and analysis of melting curves

For carrying out detection of modified nucleotides by melting of duplexes formed RNA and two probes with fluoroform and tusitala fluorescence, and sang and danced these steps:

Mixed in dies with an optically transparent cover: 4 pmol of total RNA; 2 pmol of the oligonucleotide with the donor fluorescence FAM; 4 pmol of the oligonucleotide with tusitala fluorescence BHQ1; 2 μl hbuffer for hybridization With (250 mm Tris-Hcl pH 8.3, 200 mm KCl) was added deionized water to 10 μl. After stirring the mixture from above was layered on a 5 ál mineral oil. The die is placed in the instrument for real-time PCR CFX96 (Bio-Rad) and were in the hybridization/melting in the following sequence: heated up to 80°C for 3 min, cooled to 20°C with a speed of 37 min, and then heated the mixture to 80°C with a speed of 0.5°/30 sec with simultaneous detection of fluorescence intensity at each step under heating.

Step 4. Analysis of the data obtained as a result of melting. In the experiment received the graphs of the level of fluorescence with temperature (melting curves, Figure 2), to the which were converted into curves derived fluorescence intensity on the temperature from the temperature differential curves, 3). The analysis was subjected to differential melting curves of the investigated duplexes, which celebrate the temperature corresponding to the peaks on the curves.

A flare-up of fluorescence, which corresponds to the denaturation of the shorter oligonucleotide, is expressed in the form of the inflection point on the melting curve, which corresponds to the peak on the differential curve. Comparison of the structure of RNA is carried out by comparing the temperatures corresponding peaks in the differential curves, and also by comparing the nature of the melting curves. If the nature of the curves and/or melting point are not the same for the control and test RNA conclude about the structure of the RNA in the area of the analyzed nucleotide.

The inventive method has several advantages over other methods of determining modifications. First, it is universal, that is suitable for the detection of various types of modifications of RNA. Secondly, the method allows to detect the presence of modifications, which do not prevent the formation of Watson-Cricova pairs between nucleotides and does not have a specific chemical properties that are extremely distinguishes the claimed method from most alternative methods. Thirdly, the method is extremely simple and fast, as it implies only the mixing of the components and further analysis in the course is not more than 2 hours. Fourth, the inventive method does not require high material cost analysis.

Thus, the claimed invention can be used for research purposes, such as the search for new enzymes responsible for the formation of the known modifications, to assess the level of modification of natural RNA in different conditions, as well as in the diagnosis of antibiotic resistance pathogens, where the method involves the detection of RNA, owned by some bacteria that are resistant, for example, to macrolide antibiotics.

1. The method of detection of the modified nucleotide in the composition of the RNA, including the reference RNA, two oligonucleotide probes of different length, complementary to the analyzed RNA, with one of the probes having a smaller length, complementary to the RNA in the region containing the analyzed nucleotide, and has a melting temperature less than the melting temperature of the second probe, one of the probes contains a molecule of the donor fluorescence and the other cositel fluorescence with the donor fluorescence and cositel fluorescence matched with the provision of the overlapping wavelength range emitted by the donor fluorescence radiation with a wavelength range of radiation absorbed by tusitala fluorescence; mixing of probes with study and control of PH is, then the obtained mixture is subjected to heating to a temperature that provides a complete denaturation of the analyzed RNA and probes, slow cooling to the temperature optimum for the formation of stable duplexes between RNA and probes, and re slow controlled heating with simultaneous irradiation and measurement of fluorescence intensity and obtain melting curves for the studied and the control RNA, and the conclusions about the presence or absence of modification in the tested nucleotide RNA are doing on the results of the comparison of the curves, and the coincidence of character curves, and/or the melting temperature make a conclusion about the presence/absence of modification in the studied RNA in the presence/absence of modification in the control RNA in the zone of hybridization of the shorter probe.

2. The method according to claim 1, characterized in that the control RNA using RNA with a known structure in the study area and analyzed RNA similar control or different from controlling the presence or absence of modifications in the test nucleotide.

3. The method according to claim 1, characterized in that the investigational and control RNA is ribosomal, or matrix, or transport.

4. The method according to claim 1, characterized in that the probes pick up from the condition of their hybridization to RNA in the immediate vicinity without prekriven who I am.

5. The method according to claim 1, characterized in that a longer probe pick with a melting point greater than the melting temperature of the second probe by more than 5°C.

6. The method according to claim 4, characterized in that the first and second probes pick up the conditions of hybridization to the RNA at a distance from each other not more than 10 nucleotides between their closest ends.

7. The method according to claim 1, characterized in that the first and second probes pick up the terms of the arrangement of molecules of the donor fluorescence and tusitala fluorescence at a distance of not more than 10 nucleotides from each other by hybridization of probes to RNA.

8. The method according to claim 1, characterized in that the molecules of the donor fluorescence and tusitala fluorescence to probe covalently attached.

9. The method according to claim 1, characterized in that the probes are composed of ribonucleotides and/or deoxyribonucleotides and/or their analogs, and may include any modifications that do not affect the study.

10. The method according to claim 1, characterized in that the mixture of probes with RNA is carried out by adding a buffer solution including the substance that supports the solution pH in the range of 6 to 8.5, and a salt of a monovalent or multivalent cation.

11. The method according to claim 10, characterized in that the mixture of probes with RNA using 2-10 pmol RNA, 2-10 pmol of Oleg the nucleotide probe, containing donor fluorescence, 2-10 pmol probe containing cositel fluorescence, 2-10 μl of 5 × buffer for hybridization containing 200-300 mm Tris-HCl pH 8.3, 150 and 250 mm KCl and deionized water to bring the sample volume up to 10-50 μl.

12. The method according to claim 11, characterized in that the mixture additionally contains mineral oil in the amount of 3-10 mm.

13. The method according to claim 1, characterized in that the heating and cooling of the mixtures is performed with a controlled speed, and the measurement of the intensity of fluorescence is carried out in the heating process.

14. The method according to claim 1, characterized in that the initial heating is carried out until the temperature of the mixtures 70-95°C, slow cooling occurs at a rate of 0.5-4°/min until a temperature of the mixture 30-10°C, re-heating of the mixtures produced with a speed of 0.5°/30 sec until the mixtures of 55-95°C.

15. The method according to claim 1, characterized in that the cooling is carried out to a temperature which is below the melting temperature of the short probe is at least 5°C.

16. The method according to claim 1, characterized in that the re-heating of the mixtures is carried out until the temperature, the value of which exceeds the melting point of the shorter probe is at least 5°C.

17. The method according to claim 1, characterized in that the resulting melting curves convert to curves is roizvodnykh fluorescence intensity on the temperature from the temperature differential curves).