Cationic oligonucleotides, automated synthesis methods thereof and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to oligonucleotide-oligocationic molecules AiBjH, which are used in molecular biology, diagnosis and therapeutic versions of use. The oligonucleotide-oligocationic molecules AiBjH can be synthesised via automated phosphorus amidite chemistry and have oligonucleotide fragments Ai and oligocationic fragments Bj. The fragment Ai is an i-dimensional oligonucleotide residue with index i from 5 to 50, in which nucleotide A is an oligomer with natural or synthetic nucleotide bases and/or pentafuranosyl groups and/or native phosphodiester bonds, as well as chemical modifications or substitutions thereof. Fragment Bj is a j-dimensional organic oligocationic fragment with index j from 1 to 50, in which B is selected from a group comprising -HPO3-R1-(X-R2n)n1-X-R3-O-, where R1, R2n and R3, identical or different, denote C1-C5 alkylene, X denotes NH or NC(NH2)2, index n1 = 2 to 20; -HPO3-R4-CH(R5X1)-R6-O-, where R4 denotes C1-C5 alkylene, R5 and R6, identical or different, denote C1-C5 alkylene, and X1 denotes a putrescine, spermidine or spermine residue.

EFFECT: high efficiency of the method.

18 cl, 14 dwg, 5 ex

 

The invention relates to cationic oligonucleotides, i.e. oligonucleotide-oligohaline molecules, also called cationic oligonucleotides in the description (regardless of their total charge), which can be synthesized Paladino on oligonucleotide synthesizer. The invention also relates to their use in molecular biology, diagnostics and therapeutic use cases.

Oligonucleotides are extremely wide application in molecular biology and diagnostics, and can be very selective class of drugs to treat a wide range of diseases.

Oligonucleotides are polyanion that show their specific activity according to hybridization with a complementary sequence, created another polyanionic nucleic acid.

As a potential drug candidates medicines) they must also be able to pass through the anionic cell membrane.

From simple electrostatic considerations we can conclude that the energy of hybridization and cell binding could be a favorable addition of cationic groups to the oligonucleotide structure.

In terms of the specified tasks have been investigated many synthetic approaches for the introduction of ammonium is or guanidinium residues in oligonucleotides: substitution in phosphate skeleton modification of the ribose or nucleic bases and terminal conjugation of the polycation. However, the specificity of hybridization, the activity of interacting with a nucleic acid enzyme, and toxicity of the metabolites in all respects applies to block-based approach, where the polycation joins in the rest of the natural oligonucleotide, as the best solution. Unfortunately, automated stepwise synthesis of conjugates of oligonucleotides with cationic peptides is still not a common practice. On the other hand, the chemical basis of conjugation pre-formed large molecular units remain challenging, particularly in the aquatic environment, where super-zwitterion create intractable problems in respect of solubility, purification and okharakterizovanie. Moreover, the application of molecular biology and diagnosis needs to be quick and unambiguous synthesis of any given sequence of bases associated with organic cation of any length.

The authors of the present invention have found that the real-time computer control the synthesis of oligonucleotide-oligohaline molecules was possible when installing tubes containing properly activated and protected oligohaline derivatives, oligonucleotides the first synthesizer, with the addition of the four natural bases.

Thus, the purpose of the invention is to obtain new cationic oligonucleotides.

Another objective of the invention is to develop automated synthesis of the above cationic oligonucleotides with high output.

Further objectives of the invention relates to the use of these cationic oligonucleotides, particularly in molecular biology, diagnostic and therapeutic methods.

The invention thus relates to a mixed oligonucleotide-oligohaline molecules that can be synthesized by automated phosphoramidite chemistry, i.e. using complex palifosfamide.

More specifically, cationic oligonucleotides AiBjH according to the invention are oligonucleotide fragments of Aiand oligohaline fragments of Bjwhere

Aiis a i-dimensional oligonucleotide residue, with index i = 5 to 50, with natural or non-natural nucleic acid bases and/or pentofuranose groups and/or native complex phosphodieterase relationships;

Bjis a j-dimensional organic aliocation fragment, with the index j = 1 to 50, where selected from the group including

-HPO3-R1-(X-R2)n1-X-R3-O-, where R1, R2and R3 identical or different, represent a lower alkylene, X represents NH or NC(NH2)2and index n1 = 2 to 20,

-HPO3-R4-CH(R5X1)-R6-O-, where R4represents the lowest alkylene, R5and R6identical or different, represent a lower alkylene, and X1represents putrescency, spermidine or Perminova the rest,

-HPO3-R7-(AA)n2-R8-O-, where R7represents the lowest alkylene and R8represents the lowest alkylen, serine, natural amerosport obtained by reduction of natural amino acids (AA)n2is a peptide containing natural amino acids with cationic side chains, such as arginine, lysine, ornithine, histidine, diaminopropionic acid, and the index of n2 = 2 to 20.

"Lower alkyl" or "lower alkylene", as used in the description and claims, primarily denotes optionally substituted linear or branched C1-C5-alkyl or-alkalinity radical, respectively.

But, for example, selected from the group consisting of deoxyribo-, RIBO-, closed (LNA) nucleotides, and their chemical modification or substitution, such as phosphorothioate (also called thiophosphate), 2'-fluoro-, 2'-O-alkyl or marker group, such as flu is rescently agent.

Mixed oligonucleotide-oligohaline molecules according to the invention have3'A5'-B-a sequence.

Other molecules according to the invention have In-3'A5sequence.

Still other molecules according to the invention have In-3'A5'-B or3'A5'-B3'A5'sequence.

This sequence is illustrated by examples of oligonucleotide-Perminova molecules having the following structure:

in which a and indices i and j such as defined above.

Molecules with And representing phosphorothioate nucleotide, especially preferred from the point of view of their biological applications, because phosphorothioate oligonucleotides are not hydrolyzed in biological fluids.

The above cationic oligonucleotides form a strong and stable complexes with them complementary sequences in the context of single-stranded replacement and even in the context of single-stranded plasmid invasion, as illustrated by the examples.

Thanks for the terminal conjugation selectivity sequence remains the same as for natural nucleotides.

Accordingly specified, cationic oligonucleotides according to the invention are of great interest for molecular biological the AI, as reagents for research and diagnostic applications, for example, PCR, real time PCR, genotyping, in situ hybridization and DNA chips.

Such applications are also covered by the invention and include the use of such oligonucleotide-oligohaline molecules, which is defined above.

In contrast to anionic cationic oligonucleotides the oligonucleotides according to the invention, as shown in the examples, spontaneously penetrate into the cytoplasm and nucleus of living cells.

Given their enhanced hybridization properties and penetration into the cell, they are also useful for therapeutic methods, such as methods using the degradation of messenger RNA directed antimuslim and short non-coding RNAS (siRNAs), exon skipping during the maturation of messenger RNA, triple helix formation with chromatin, chromatin-stranded invasion (gene correction)...

The invention thus relates also to pharmaceutical compositions containing an effective amount of the oligonucleotide-oligohaline molecules such as described above, in combination with a pharmaceutically acceptable carrier.

The invention also relates to a method of treatment comprising applying an effective amount of the oligonucleotide-oligohaline molecules such as described above, in combination with f is rmaceuticals acceptable carrier.

The above mixed oligonucleotide-oligohaline molecules predominantly synthesized Paladino in oligonucleotide synthesizer, phosphoramidites way, according to the method, including

- placing test tubes containing activated and protected aliocation In oligonucleotide synthesizer, adding a tube of oligonucleotides And such as described above, or Vice versa,

- stop synthesis is achieved when the desired length,

- cleavage of the oligomer from the solid media

- remove the protective groups.

The invention is directly related to the synthesis for design aliocation repeating unit Century. For this purpose can be used the following phosphoramidite reagents

P(OR9)(N(R10)2)-O-R1-(X-R2)n1-X-R3-O-Prot, where R1, R2, R3and index n1 such as defined above, X represents an appropriately protected NH or NC(NH2)2, R9represents-CH2CH2CN or lower alkyl, R10represents lower alkyl, or-N(R10)2is a pyrolidine, piperidine or morpholino group, and Prot is a protective group used in oligonucleotide synthesis, such as DMT, MMT;

P(OR9)(N(R10)2)O-R 4-CH(R5X1)-R6-O-Prot, where R4, R5and R6constitute the lower alkylene, X1represents an appropriately protected putrescine, spermidine or spermine, R9and R10such as described above;

P(OR9)(N(R10)2)-O-R7-(AA)n2-R8-O-Prot, where R7, R8, R9, R10, n2 and Prot such as described above, (AA)n2is a peptide containing natural amino acids with appropriately protected cationic side chains, such as arginine, lysine, ornithine, histidine, diaminopropionic acid, and the index of n2 = 2 to 20.

"Appropriately protected NH or NC(NH2)2" means amino or guanidine residue, respectively, are present protective groups to make these functional groups inert under the conditions of chemical reactions, which is exposed to the reagent.

Such protective groups are, for example, talimena (RNT), trifurcata, allyloxycarbonyl (Alloc), benzyloxycarbonyl (CBZ), chlorantraniliprole, tert-butyloxycarbonyl (Vos), fluorenylmethoxycarbonyl (Fmoc) and isonicotinamide (i-Noc) groups.

According to a variant of the invention, the stepwise synthesis of the oligonucleotide sequence proceeds stepwise Sint the zoom aliocation component for producing compounds having the sequence (3'A5'-B).

According to another variant embodiment of the invention is a reverse order of stages, with sequential synthesis aliocation component, which continues sequential synthesis of the oligonucleotide sequence to obtain compounds with a sequence of (3'A5').

According to another further variant embodiment of the invention are synthesized mixed sequence.

In particular, oligonucleotide sequences that putatively capped at both ends (In-3'A5'-In), can be resistant to ectonucleoside in biological fluids, and the sequence with cationic interrupt (3'A5'In3'A5'allow to position the adjacent ("vicinal") nucleic acid sequences.

The use of natural amines, such as spermine, or peptides, such as oligoaniline, eliminates the potential toxicity of metabolites. Spermin actually present in millimolar concentrations in cells, and its end alkylation harmless. Moreover, the base peptide sequence is present in many nuclear proteins.

Activated and protected aliocation predominantly obtained by protection of the amino groups polyamine, with subsequent α,ω-bisher what kalkiliya, leading to dilam compatible with oligonucleotide synthesis.

Classical chemical approach DMT and phosphoramidites capacity circuit preferably used in combination with the use of sensitive grounds protective groups FA.

Chemically protected diols are new products and are included in the scope of the invention.

The invention in particular relates to intermediate products selected from the group including

P(OR9)(N(R10)2)-O-R1-(X-R2)n1XR3-O-Prot, where R1, R2, R3and index n1 such as defined above, X represents an appropriately protected NH or NC(NH2)2, R9represents-CH2CH2CN, or lower alkyl, R10represents lower alkyl, or-N(R10)2is a pyrolidine, piperidine or morpholino group, and Prot is a protective group used in oligonucleotide synthesis, such as DMT, MMT;

P(OR9)(N(R10)2)-O-R4-CH(R5X1)-R6-O-Prot, where R4, R5, R6constitute the lower alkylene, X1represents an appropriately protected putrescine, spermidine or spermine, R9and R10such as described above;

P(OR9)(N(R10)2)-O-R7-(AA)n2-R8-O-Prot, where R 7, R8, R9, R10, n2 and Prot such as described above, (AA)n2is a peptide containing natural amino acids with appropriately protected cationic side chains, such as arginine, lysine, ornithine, histidine, diaminopropionic acid, and the index of n2 = 2 to 20.

Other characteristics and advantages of the invention are given below. In particular, the synthesis decameric oligonucleotide sequences (A10with spermine (S), in the following denoted As10Snwill be shown as an illustrative example, without limitation of the invention. In the examples, it will be described Fig.1-14, which represent, respectively:

figure 1 - HPLC analysis of cationic oligonucleotides N10Sn(n=1-2) column with reversed phase,

figure 2 - analysis of HPLC purified oligonucleotides N10Sn(n=1-6) on the anion-exchange column,

figure 3 - analysis of the electrophoretic mobility of N10Sn(n=1-6) in polyacrylamide gel electrophoresis,

4 is a spontaneous substitution of N10fragments of N10·10at different temperatures,

5 is a chain of substitution between the N10and N10Snas manifested during electrophoresis polyamide gel

6 is a melting temperature of duplexes N10Sn·10(where is the FDS is the first nucleotide, complementary N),

7 - comparative results of the melting temperature of the duplexes formed by oligonucleotides N10Sn(n=0-6)5'GTGGCATCGC3'and5'GTGGCGTCGC3',

Fig - analysis by the method of mass spectrometry with ionization by sputtering in an electric field (electrospray, ES-MS) of purified N10Sn(n=1-6) oligonucleotides,

Fig.9 - HPLC detects phosphorothioate oligonucleotides N12S11F (9A) and N12S2F (9V),

figure 10 - mass spectra of MALDI-TOF MS N12S2F (10A) and (N12S11F (10V),

11 - HPLC detects N14S4F (11A) and (N20S5F (11B), respectively,

Fig - mass spectra of MALDI-TOF MS N14S4F (12A) and (N20S5F (12V),

Fig - stranded invasion plasmid pGL2 and pGL3 oligonucleotide-N14SnF (13A) and (N20SnF (13C).

figa and 14C - penetration of cationic oligonucleotide F-S18N19HeLa cells.

Example 1: Synthesis phosphoramidites sperminator Cinchona

Phosphoramidite 1 based spermine was synthesized from spermine, as shown in the following scheme 1:

(Mes = 2,4,6-trimetilfenil; TBDMS = tert-butyldimethylsilyl; TFA = CF3CO-; DMT = 4,4'-dimethoxytrityl)

Tetrakis(mesitylenesulfonyl)spermin 2, prepared from spermine, subjected bialkali is to Finance with the formation of intermediate 3. After complete removal of the protective groups with intermediate 3 in acidic conditions crude tetrahydroborate bis(C4-HE)spermine 4 fully protected by trifluoroacetic anhydride in pyridine, followed by two terminal ester groups in the intermediate 5 hydrolyzed in neutral conditions with the formation of diol 6. Monomethylamine intermediate 5 was performed in the statistical mode using one molar equivalent of the reagent DMTCl (dimethoxytrityl) with the formation of intermediate 7 with the release of 43%. Unreacted diol 6 and bistricioara compound 8 was recovered and brought to a new equilibrium in mild acidic conditions (triperoxonane acid in dichloromethane) with the formation of intermediate 7. Fosfaurilirovaniem intermediate 7 was obtained the desired phosphoramidite 1.

N1N4N9N12-tetrakis(mesitylenesulfonyl)spermine (2): The compound was prepared according to: Bergeron et al., J. Med. Chem., 2001, volume 44, pages 232-244.

N1N12bis[4-(tert-butyldimethylsilyloxy)butyl]-N1N4N9N12-tetrakis(mesitylenesulfonyl)spermine (3): To a solution of compound 2 (9,31 g, 10.0 mmol) in dimethylformamide (DMF) (20 ml) under stirring in nitrogen atmosphere at a temperature of 0OC portions was added sodium hydride (60%, 1.0 g, 25 mmol). After stirring at room te is the temperature for 30 min to one portion was added tert-butyl(4-iodobutane)dimethylsilane (7,86 g, 25 mmol). The mixture was stirred over night at room temperature and then was diluted with water (100 ml) and was extracted with dichloromethane (100 ml). The organic phase was separated, and the aqueous phase was extracted three times with dichloromethane (50 ml). The combined organic phases were washed with a solution of NaHCO3(1 M) and then dried over MgSO4. After evaporation of the pasty residue was purified using flash chromatography using a mixture of AcOEt:cyclohexane 1:4 as eluent. The fractions containing product 3, was evaporated with the formation of a viscous oil, which was further washed with cold pentane to remove mobile (chromatogram) impurities, and then dried under vacuum to education becomes 9.97 g (76%) of product 3 as an oil: TLC (AcOEt/cyclohexane 1:4): Rf=0,28. IR (KRS-5): 2937, 1604, 1471, 1320, 1151, 1101, 838, 777, 657, 578 cm-1.1H-NMR (300 MHz, CDCl3): δ=-0,01 (s, 12H), of 0.85 (s, 18H), 1,20-of 1.45 (m, 12H), of 1.62 (m, 4H), of 2.28 (s, 6H), to 2.29 (s, 6H), 2,53 (s, 12H), of 2.54 (s, 12H), 2,90-3,10 (m, 16H), 3,42 (t, J=6,1 Hz, 4H), 6,91 (s, 4H), 6,92 (s, 4H).13C-NMR (75 MHz, CDCl3): δ=4,7, 18,9, 21,6, 23,4, 23,5, 24,1, 24,9, 25,7, 26,6, 30,4, 43,5, 43,6, 45,6, 45,7, 62,9, 132,59, 132,64, 133,8, 140,7, 143,0, 143,1. MS-ESI (MeOH): m/z=1325,85 [M+Na]+, 1303,83 [M+H]+. C66H110N4O10S4Si2(Mw=1304,03) calculated C 60,79, H 8,50, N 4,30, S 9,84; found C 60,74, H 8,55, N 4,21, S 9,63.

Tetrahydroborate N1N12bis(4-hydroxybutyl)spermine (4): To a solution of compound 3 (9,87 g, EUR 7.57 shall mol) and phenol (29.0 g, 0.31 mol, 40 equivalents) in CH2Cl2(80 ml) was added dropwise a solution of bromovalerate in acetic acid (33%by weight solution, 80 ml, 1.4 mol). The reaction mixture was stirred over night at room temperature. While cooling in an ice bath was added with stirring cold water (100 ml). The organic layer was separated and was extracted three times with water (20 ml). The combined aqueous layers washed five times CH2Cl2(30 ml) and evaporated to dryness. The resulting crude solid residue suspended in ether, triturated with a spatula, and the supernatant ether layer was decanted. These operations were repeated (five times)until they received a suspension of solids. After evaporation and drying in vacuum were obtained compound 4 in the form of a solid (5.32 g). The resulting crude material was used without further purification:1H-NMR (300 MHz, D2O): δ=1,75-2,10 (m, N), and 2.27 (m, 4H), 3,15-to 3.35 (m, N), 3,76 (t, J=and 12.2 Hz, 4H).13C-NMR (75 MHz, D2O): δ=22,9, 23,2, 23,4, 29,0, 45,0, 45,2, 47,7, 48,3, 61,5. MS-ESI (Meon): m/z=347,39 [M+H]+.

N1N12bis(4-(triptoreline)butyl)-N1N4N9N12-tetrakis(TRIFLUOROACETYL)spermine (5) (from compound 4 with FA2O/NEt3): To a suspension of compound 4 (5,3 g, 7.6 mmol) in CH2Cl2(50 ml) in one portion was added triethylamine (11.5 g, 114 mmol, 15 equivalents). The mixture was cooled in an ice bath and preparemessage in an atmosphere of nitrogen was added dropwise triperoxonane anhydride (19,1 g, 90,9 mmol, 12 equivalents). The mixture was stirred at room temperature for 3.5 hours, After cooling in an ice bath, the resulting solution was washed three times with cold water (20 ml), dried over MgSO4and then was evaporated with the formation of an oily residue (11,7 g), which as a by-product of the specified reaction contains (TFA)2C=CH-NEt2(reference: Schreber, S. L., Tetrahedron Lett., 1980, volume 21, page 1027). The obtained product was removed by two consecutive operations flash chromatography (eluent in gradient from 1:1 to 60:40 AcOEt:cyclohexane, and then 5-10% Et2O/CH2Cl2with the formation of product 5 (5,59 g, 81%) as an oil: TLC (AcOEt/cyclohexane 1:1): Rf=0,25. IR (KRS-5): 2955, 1789, 1690, 1467, 1352, 1197, 1147, 759, 731, 692 cm-1.1H-NMR (300 MHz, CDCl3): δ=1,52 e 2.06 (m, 16H), 3,33-to 3.49 (m, 16H), to 3.38 (m, 4H).13C-NMR (75 MHz, CDCl3): The spectrum is complicated by the presence of rotational isomerism four amide groups. Describe only the resonance signals of high intensity, as follows: δ=23,3, 23,9, 24,1, 24,8, 25,3, 25,6, 26,0, 26,55, 26,61, 44,4, 44,8, 45,7, 46,1, 46,4, 47,3, 48,0, 56,6, 67,3, 67,5, 116,6 (kV, J=288 Hz), 156,9, 157,4, 157,8, 158,6.

N1N12bis(4-hydroxybutyl)-N1N4N9N12-tetrakis(TRIFLUOROACETYL)spermine (6): To a solution of compound 5 (of 5.39 g of 5.84 mmol) in Meon (50 ml) in one portion was added NaHCO3(0.1 g, solid), and the resulting suspension was stirred for 2 h the owls at room temperature. After evaporation of the oily residue was dissolved in CH2Cl2(with formation of a suspension a quantity of fibrous NaHCO3) and was purified using flash chromatography, washing out with a mixture of 5-10% MeOH/CH2Cl2education 3,61 g (85%) of the product 6 in the form of an oil: TLC (5% MeOH/CH2Cl2): Rf=0,14. (10% MeOH/CH2Cl2): Rf=0,45.1H-NMR (300 MHz, CDCl3): δ=1,51-2,02 (m, N), 3,33-3,51 (m, N), 3,68 (m, 4H). MS-ESI (Meon): m/z=753,33 [M+Na]+. With26H38F12N4O6·H2O (Mw=748,60) calculated 41,72, N 5,39, N Of 7.48, F 30,45; found C 41,97, H 5,26, N 7,37, F 30,14.

The product 6 from compound 4 (TFA2O/pyridine, then NaHCO3): To a suspension of compound 4 (15.3 g, of 22.8 mmol) in CH2Cl2(100 ml) and pyridine (44 ml, 0.54 mol) under cooling in an ice bath and stirring in nitrogen atmosphere was added dropwise triperoxonane anhydride (46 ml, 0.33 mol). The mixture was stirred at room temperature for 3 hours. Excess triperoxonane anhydride was decomposed by the addition of cold water (100 ml) under cooling in an ice bath, and then the resulting solution was extracted with dichloromethane CH2Cl2(four times, 100 ml + 50 ml + 25 ml ×2). The combined extracts were washed with cold water (50 ml ×3), dried over MgSO4and then was evaporated with the formation of the crude product 5 (19,4 g, 92%) as oil. The oil obtained RA is tarali in the Meon (100 ml). Added NaHCO3(solid, 0.1 g), and the suspension was stirred over night. After evaporation of the solvent the residue was purified flash chromatography using a mixture of 5-7% Meon:CH2Cl2as eluent with the formation of 10.1 g (61%) of the product 6 in the form of oil.

N1-[4-(dimethoxytrityl)butyl]-N12-(4-hydroxybutyl)-N1N4N9N12-tetrakis(TRIFLUOROACETYL)spermine (7): To a solution of compound 6 (1,46 g, 2.00 mmol) in pyridine (3 ml) was added DMTCl (757 mg, of 2.23 mmol)using 1 ml of pyridine for complete rinsing. The reaction mixture was stirred for 4 hours at room temperature in a nitrogen atmosphere, and then the pyridine was removed by repeated perevarivanii with toluene. The residue was purified by two sequential operations flash chromatography (eluent 2-5% Meon/CH2Cl2and then 10-15% acetone/CH2Cl2with the formation of the product 7 (879 mg, 43%) as a foam, and bis-DMT-protected derivative 8 (648 mg, 24%). Also return the original diol 6 (350 mg, 24%). Data for 7: TLC (acetone/CH2Cl21:9): Rf=0,20.1H-NMR (300 MHz, CDCl3): δ=1,51-2,03 (m, 17H), 3,11 (m, 2H), 3,32-3,51 (m, N), 3,71 (m, 2H), 3,81 (C, 6N), at 6.84 (m, 4H), 7,19-7,46 (m, N). MS-ESI (Meon): m/z=1055,52 [M+Na]+. C47H56F12N4O8(Mw=1032,95) calculated 54,65, N 5,46, N 5,42, F 22,07; found C 54,46, H 5,58, lower than the 5.37 N, F 21,63.

Connection (7) of the diol (6) and bis-DMT-protected proizvodnjo (8): To a solution of compound 6 (1.4 g, 1.9 mmol) and compound 8 (2.5 g, 1.9 mmol) in CH2Cl2added triperoxonane acid (50 μl, 0.6 mmol) and stirred at room temperature for 30 minutes. Solution three times washed with 1 m solution of Na2CO3, dried over MgSO4and was evaporated. The residue was separated by flash chromatography (column diameter: 50 mm, height SiO2: 15 cm) using successively eluents 5% AcOEt/CH2Cl2(750 ml), 33% AcOEt/CH2Cl2(500 ml), 7% Meon/CH2Cl2(500 ml) and 10% Meon/CH2Cl2(500 ml) with the formation of 8 product (1.1 g), 7 product (1.2 g) and 6 (1.3 g).

Phosphoramidite based spermine (1): To a solution of compound 7 (844 mg, 817 mmol) and triethylamine (230 μl, of 1.65 mmol, 2 equivalent) in CH2Cl2(4 ml) was added 2-cyanoethyl-(N,N-diisopropylamino)chlorophosphite (205 μl, of 0.92 mmol, 1.1 equivalent), and the mixture was stirred in nitrogen atmosphere at room temperature for 40 minutes. The reaction mixture was passed through a column of SiO2(diameter: 20 mm, height: 15 cm), saturated NEt3(1% of NEt3in CH2Cl2:cyclohexane 1:2, 400 ml) using a mixture of 1% of NEt3in CH2Cl2:cyclohexane 1:2 (125 ml) and then 1% of the NEt3in CH2Cl2:cyclohexane 1:1 (100 ml) with the formation of product 1 (735 mg, 73%) as an oil:1H-NMR (200 MHz, CDCl3): δ=1,13-of 1.35 (m, N)and 1.51-to 2.06 (m, N), to 2.66 (t, J=6.4 Hz, 2H), 3,11 (who, 2H), 3,32-3,98 (m, 20N), 3,81 (C, 6N), at 6.84 (m, 4H), 7,15-7,51 (m, N).31P-NMR (81 MHz, CDCl3): 148,06, 148,13, 148,19, 148,3 (the splitting due to rotational isomerism amide groups).

Example 2: Synthesis, purification and okharakterizovanie decameric oligonucleotides having the formula

These oligonucleotides will be further denoted N10Sn(N10= oligonucleotide fragment; S = Perminova the residue, and the index n=1-6).

Automated synthesis: A series decameric oligonucleotides with identical sequences of N10=3'CACCGTAGCG5'connected with the growing number Perminova residues S, synthesized using standard chemical solid-phase approach cyanomethylphosphonate method Expedite synthesizer DNA, according to the following scheme:

with nuke as a last N-fragment according to the classical oligonucleotide synthesis.

The reagents used for automated DNA synthesis were purchased in the firm Glen Research (Eurogentec).

During automated synthesis used a standard 1-micropoly cycle combination, except combinations sperminator of phosphoramidite 1, which was held with prolonged time combinations (15 minutes) and using a slightly more towards the centered solution phosphoramidite (90 mg amidite in 1 ml acetonitrile).

Triteleia fractions were collected, diluted and analyzed in a spectrophotometer to determine the output sequential combinations.

The outputs of the combination of the four natural nucleotides exceeded 97%, whereas the outputs of the combination sperminator of phosphoramidite was between 90 and 96% in the above-mentioned combination of conditions.

In all cases we used a variant of the DMT-ON (ON = oligonucleotide (oligonucleotide with dimethoxytrityl protection), keeping the 5'-terminal DMT group neotdalennyh on the oligomers for the purposes of purification and identification.

Processing after synthesisAfter the automated synthesis was performed cleavage from the solid media and the complete removal of the protective groups of the oligomers using standard conditions (treatment with concentrated aqueous ammonia for 90 minutes at room temperature to detach and then over night at a temperature of 55ºC for removing the protective groups).

Clean: The first two anionic oligonucleotide N10S1and N10S2were initially cleaned in a DMT-protected condition by standard HPLC method column with reversed-phase media Nucleosil C-18 (company Macherey-Nagel, size 10×250 mm) with a linear gradient of acetonitrile (5-35% for 20 minutes) in 20 mm solution of ammonium acetate (pH 7). With purified oligonucleotides were then removed trailing protection processing see what sue Asón/N 2O=4/1 (500 ml) at room temperature for 20 minutes. After dilution with water (5 ml) the resulting DMT-OH was removed by extraction with ether (3×2 ml)and the aqueous phase was concentrated to obtain oligomers.

Data HPLC of oligonucleotides N10S1and N10S2shown in figure 1; column reversed-phase media Nucleosil C-18 (company Macherey-Nagel, a 4.6×250 mm) with a linear gradient of acetonitrile (5-35% for 20 minutes) in 20 mm solution of ammonium acetate (pH 7): a) N10S1, raw, DMT-protected oligonucleotide (DMT-ON); (b) N10S1cleared; (C) N10S2, raw, DMT-protected oligonucleotide (DMT-ON); (d) N10S2cleaned. *Benzamid; **Shortened sequence.

Neutral oligomer N10S3and cationic oligomers of N10S4N10S5and N10S6(DMT protective group, or without it) were purified using columns Poly-Pak IITM(Glen Research/Eurogentec) according to the instructions provided by the manufacturer, except for the final elution of the oligonucleotide, which was conducted with a mixture of acetonitrile/concentrated aqueous ammonia/water (20:4:80). The fractions containing the oligonucleotides could be detected by TLC. After collecting the fractions, the solvents were removed by lyophilization. The resulting oligomers were generally dirty be what samida. It was removed by extraction with ether (three times) after dissolution in a dilute solution of aqueous ammonia (50 mm). Purified oligonucleotides were dissolved in a dilute solution of aqueous ammonia (50 mm), and their concentration was determined using the following extinction coefficient (260 nm, mol-1DM3cm-1):

ε=(15,4NAnd+11,5NG+7,4NC+8,7NT)×0,9×103.

Data HPLC purified oligonucleotides is shown in figure 2: anion exchange column (Dionex PA-100, 9×250 mm) with a linear gradient of NaCl (concentrations from 100 mm to 350 mm for 10 minutes)/NaOH, 25 mm (pH 12,4): a) N10S1b) N10S2with N10S3d) N10S4e) N10S5f) N10S6.

Thanks to the use of chemical conjugation approach each polyamine comes with a phosphate group, thereby making the system more cationic charges. Seven oligonucleotides, (N10Sn)3n-9n=0...6, with the total charges -9, -6, -3, 0, +3, +6, +9, being fully ionized, thus are available in amounts ranging from 80 to 250 nanomoles.

Electrophoretic mobility

Their migration in an electric field with the value of pH 7 was studied by the method of polyacrylamide gel electrophoresis with manifestation on the spot silver mirror. Compounds (0.5 nmol) at 0 μl of buffer media (10 mm HEPES buffer with pH 7.4, 150 mm NaCl, glycerol) were placed in sedentarise polyacrylamide gel (15% in TAE-buffer, pH 7). Electrophoresis was carried out at 5 V/cm for 17 hours at a temperature of 4ºC. Silver staining was performed according to Rabilloud et al., Electrophoresis, 1987, volume 9, pages 288-291. The results are shown in figure 3. Oligonucleotide N10(track 1) without spermine quickly moved to the anode and showed only a weak silver staining in conditions where identified oligonucleotides containing polyamine.

Spontaneous substitution10fragments N10·With10

Oligonucleotide10(where C is a nucleotide complementary to N) (50 pmol 500 pmol) was added to a solution of fluorescent duplex N10·C10* (50 pmol in 10 mm HEPES buffer, pH 7.4, 150 mm NaCl). Mixtures were incubated for 4 hours at a temperature of 37º, 20 or 10 º C and placed in sedentarise polyacrylamide gel (15% in TAE-buffer, pH 7). The electrophoresis was carried out at a temperature of 4ºC for 17 hours at 5 In/see10*-fluorescence were detected by scanning the gel using a device Typhoon 8600 Imager. As shown by the results shown in figure 4, the spontaneous substitution of N10fragments of N10·10is not significant at a temperature of 10 º C.

Single-stranded substitution between N10and N10S/i> n

The ability to single-stranded replacement of N10Snin relation to the natural duplex N10·C10tested in physiological solution.

Spermine conjugates of N10Sn(50 or 500 pmol) was added to a solution of fluorescent duplex N10·C10* (50 pmol in 10 mm HEPES buffer, pH 7.4, 150 mm NaCl). Mixtures were incubated for 4 hours at a temperature of 10 º C, and placed in sedentarise polyacrylamide gel (15% in TAE-buffer, pH 7). The electrophoresis was carried out at a temperature of 4ºC for 17 hours at 5/, see Fluorescence were detected by scanning the gel using a device Typhoon 8600 Imager.

The conjugation of spermine had a strong influence on the reaction single-replacement, as shown in figure 5. The band corresponding to N10·C10*became weaker with increasing number Perminova balances competing in N10Snin favor of less rolling less anionic complex of N10Sn·C10*. This effect was particularly pronounced for N10S3that is , for conjugates that were not carrying a formal negative charge. Indeed, spermine binds duplex DNA structures by forming miaocheng network of NH2+-bidentate hydrogen bonds in the minor groove of the DNA helix, thereby JV is mabstoa binding of N 10Sncompared with N10. Besides and additionally favorable kinetic factor can act when single-stranded substitution occurs in the pre-formed electrostatic complex (N10Sn)3n-9/(N10·C10)18-that can occur for n>3.

The melting temperature of duplexes N10Sn·C10

Stability of double-stranded nucleic acids were compared by measuring their melting temperature, i.e. the temperature at which complementary chain cooperative diverge. This was recorded at a wavelength of 260 nm optical density (D) solutions of N10Sn·C10relative to the temperature T.

The melting temperature TPLmeasured in 10 mm HEPES buffer, pH 7.4 (black line, diamonds) and 10 mm HEPES buffer, pH 7.4 + 150 mm NaCl (grey line, circles). The melting profiles of all duplexes (3.75 nmol in 1 ml buffer) was observed using a spectrophotometer CARY 4000 units with temperature control, during the gradual heating of the samples (1OC/min), at the same time recording their absorption at the wavelength of 260 nm. Melting of duplexes was manifested in hyperchromic shift, and TPLrepresents the temperature at which the curve first derivative dOD/dT=f(T) reaches its maximum. Raza is taty shown in figure 5.

Natural duplex melts at TPL=30 º C in 10 mm HEPES buffer with pH 7.4 (figure 5). Algae growing number Perminova residues leads to a considerable improvement of the values of TPL. N10S6·C10melts at TPL=75,2C, almost 45 º C higher than the natural duplex. Curve TPL=f(n) reveals a sigmoidal shape with a break for neutral oligonucleotide N10S3.

The melting temperature were also recorded in physiological solution. Curve TPL=f(n) is more upright and, interestingly, previous crosses the curve at the point for N10S3. Thus, for n<3 as oligonucleotides N10Snand oligonucleotides With10are anionic and are repelled from each other in the duplex; increasing the concentration of salt screens the repulsive force, thereby increasing the value of TPL. For n>3 N10Snbecomes cationic and attracts With10; here due to salt electrostatic shielding reduces stability.

For neutral N10S3the stability of the duplex does not depend on the salt concentration.

Comparison of the melting temperatures of the duplexes formed N10Sn(n=0-6)5'GTGGCATCGC3'and5'GTGGCGTCGC3'

p> It was tested discernment erroneous pairing ("mismatch") single base pair oligonucleotide-Spiridovich conjugates. Within the context of sequences With10=5'GTGGCATCGC3'data from the literature recommend the centrally localized conversion of the "A-G" as the most stringent criteria.

The melting temperature TPLmeasured in 10 mm HEPES buffer with pH 7.4 + 150 mm NaCl. The melting profiles of all duplexes (3.75 nmol in 1 ml buffer) was observed using a spectrophotometer CARY 4000 units with temperature control, during the gradual heating of the samples (1OC/min), at the same time recording their absorption at the wavelength of 260 nm. TPLrepresents the temperature at which the curve first derivative dOD/dT=f(T) reaches its maximum. The results are shown in Fig.7 (diamonds correspond to the5'GTGGCATCGC3'and the triangles correspond to the5'GTGGCGTCGC3').

The transition temperature of the natural duplex N10·C10in 150 mm NaCl fell from 50,6º to 42,9º, i.e. DTPL=7,7 º C, when present mismatch. In principle, the increase in stability due to non-specific electrostatic forces at the end of the conjugation should not diminish the specificity of base pairs, which is expressed as ΔΔG. It is observed in reality, as complementary mismatched target oligonucleotides showed quasiparallel curves T PL=f(n) with an average value of ΔTPL=7,9º.

Analysis of the ES-MS of purified oligonucleotides N10Sn

The oligonucleotides were dissolved in 50% (by volume) aqueous acetonitrile containing 1% of triethylamine at a final concentration of 5×10-5M. Aliquots of 100 ml were injected into the ion source of the mass spectrometer Mariner 5155 company Applied Biosystems at a flow rate of 5 ml/min. and the Results are shown in Fig (inserts: spectra deconvolution): a) N10S1b) N10S2with N10S3d) N10S4e) N10S5f) N10S6. Ionization of neutral and cationic oligomers of N10S3-6became more difficult, and required the accumulation of multiple spectra to obtain acceptable signal-to-noise ratio.

Example 3: Synthesis, purification and characterization of a 12-dimensional thiophosphate oligonucleotides having the formula

These oligonucleotides will be further denoted N12SnF (N = 12-dimensional thiophosphate-oligonucleotide fragment; S = Perminova the residue, and the index n = 2 to 11; F = fluorescein conjugated with thymine).

Automated synthesis:12-dimensional thiophosphate-oligonucleotides with a sequence of N12=3'GCGACTCATGAA5'connected with the number Perminova residues S from two to 11, Sintesi the Wali using solid-phase cyanomethylphosphonate chemical approach to Expedite synthesizer DNA. Phosphoramidite UltraMILD CE and media UltraMILD (Glen Research/Eurogentec) was used in order to avoid cleavage of the oligomer during processing after synthesis. Standard sulfurylase reagent (Glen Research/Eurogentec) was used to create phosphorothioate ligaments in 12-dimensional oligonucleotide structure. The fluorescein-dT-phosphoramidite (Glen Research/Eurogentec) was used for labeling the 5'-end. The combination Perminova of phosphoramidites were performed using combination techniques described in example 2.

Triteleia fractions were collected, diluted and analyzed in a spectrophotometer to determine the output sequential combinations.

In all cases we used a variant of the DMT-ON, keeping the 5'-terminal DMT group neotdalennyh on the oligomers for the purposes of purification and identification.

Processing after synthesis:After automated synthesis cleavage from the solid media and the complete removal of the protective groups of the oligomers was performed by treatment with concentrated aqueous ammonia overnight at room temperature.

Cleaning:DMT-ON connection N12S2F and N12S11F was purified using columns Poly-Pak IITM(Glen Research/Eurogentec) according to the instructions provided by the manufacturer.

Purified oligonucleotides N12SnF (n=2, 11) were analyzed by anion-exchange column (SAX1000-8) in aqueous alkaline conditions (100 mm ammonia, pH 1) with a gradient of NaCl (0.75 To 2.5 M within 20 minutes). The peaks of HPLC is shown in Fig.9 (A: N12S11F,: N12S2F).

Analysis of MALDI-TOF MS of purified oligonucleotides

The oligonucleotides were dissolved in 500 μl of demineralized water. Sample and NRA-matrix (gidroksipropanova acid) were mixed together on the plate. After crystallization, the sample was analyzed on the instrument BRUKER MS wall-mounted. The results are shown in figa: N12S2F calculated 5460 found 5459 (top), and figw: N12S11F calculated 9135 found 9125 (bottom).

Example 4: single-Stranded invasion plasmid DNA from 14-gauge and 20-dimensional fluorescent oligonucleotide -

The connections shown above, will be denoted as N14SnF (N = oligonucleotide fragment; S = Perminova the residue with n=2-4; F = fluorescently balance), and as N20SnF (N = oligonucleotide fragment; S = Perminova the residue with n=3-5; F = fluorescently balance).

These fluorescent oligonucleotides were synthesized according to the procedure described in example 2. 5'-fluorescein-phosphoramidite (Glen Research/Eurogentec) was used for labeling the 5'-end. Analytical peaks of the HPLC and MALDI-TOF MS for the most substituted compounds N14S4F and N20S5F is shown at 11 and 12 as evidence of the purity and structure(N 14S4F calculated 6470 found 6478; N20S5F calculated 8813 found 8815), respectively.

Oligonucleotide sequence of N14SnF and N20SnF were selected within the sequence of the gene luciferase control plasmid pGL3 (Promega). To assess the specificity of the sequence in single-stranded invasion applied control plasmid pGL2 (Promega). The sequence of GL2-luciferase is 95% identical to GL3, and the sequence of the target for N14SnF and N20SnF, contain, respectively, one or two mismatch.

The ability of N14SnF and N20SnF to single-stranded invasion plasmid pGL3, but not in pGL2, experienced in physiological solution and temperature.

Fluorescent conjugates of N14SnF and N20SnF (8,65 pmol) was added to a solution of plasmids (1.5 mcg, 0.43 pmol in 10 mm HEPES buffer with pH 7.4, 150 mm NaCl). Mixtures were incubated for 24 hours at a temperature of 37º and was placed in the agarose gel (1.3% in TAE-buffer with pH 7.4). Electrophoresis was carried out at room temperature for 45 minutes after the detection of green fluorescence of fluorescein by scanning the gel using a device Typhoon 8600 Imager. The picture of the red fluorescence of the gel was photographed on a UV-UV-transilluminator with subsequent incubation for 15 minutes the solution ethidiumbromid. The results are shown in Fig.

Red and green fluorescence are proof of double-stranded binding of plasmid DNA and fluorescent oligonucleotide, respectively. Their colocalization with pGL3, but not with pGL2 is thus proof single strand invasion. Connection N14S3F and N20SnF showed fuzzy strip of green fluorescence associated with the plasmid, when were incubated with pGL3, but not with pGL2.

Example 5: Penetration of cationic oligonucleotides in cells

HeLa cells grown in 10% (by volume) minimum nutrient medium (MEM)containing fetal calf serum were placed in 4-hole chambered Cup of borosilicate glass Lab-Tek at 50-60×103cells/well one day prior to the experiment. Complete medium for cultivation was replaced by 0.5 ml of MEM-a medium without serum. 5'-cationic conjugated with fluorescein oligonucleotide F-S18N19(where N19represents TCGAAGTACTCAGCGTAAG) composition was prepared in sterile phosphate-buffered saline (PBS). It was added to cells to a final concentration of 2 μm. Four hours later the medium was replaced by 1 ml of fresh medium containing serum. The first image was shot on a fluorescent microscope (Zeiss axiovert 25, equipped with a blue interference filter (FIT) (figa, left). All tile and began to fluoresce with some fluorescence, located in intracellular vacuoles and, most importantly, also common in the cytoplasm and nucleus. After 24 hours the medium was replaced by 1 ml of MEM-a medium that does not contain phenol red. Added propidium iodide (1 mm in water) to a final concentration of 10 μm. Ten minutes later filmed the second picture, showing the most healthy cells, not including propidium, which was still fluorescently (FIGU, right). Control cells that were incubated under similar conditions with the oligonucleotide F-N19, showed no fluorescence.

Thus, the invention represents a generic automated synthesis of cationic oligonucleotides that form a strong and stable complexes with them complementary sequence even in the context of single-stranded invasion. Thanks for the terminal conjugation selectivity sequence remains high, as for natural oligonucleotides. Moreover, due to their cationic nature, delivery inside cells does not require the formation of a complex with cationic molecules media. Taken together, these characteristics make oligonucleotide-oligohaline conjugates attractive alternative to oligonucleotides for molecular biology, diagnostics, and therapeutic applications.

1. Oligonucleotide-oligohaline is olekuly A iBjH, which can be synthesized by automated phosphoramidite chemistry with oligonucleotide fragments of Aiand oligohaline fragments of Bjwhere
Aiis a i-dimensional oligonucleotide residue with index i = 5 to 50, with natural and non-natural nucleic acid bases and/or pentofuranose groups and/or native complex phosphodieterase links and their chemical modification or substitution
Bjis a j-dimensional organic aliocation fragment with index j = 1 to 50, where selected from the group including
-HPO3-R1-(X-R2)n1-X-R3-O-, where R1, R2and R3identical or different, represent a C1-C5alkylene, X represents NH or NC(NH2)2and index n1 = 2 to 20,
-NRA3-R4-CH(R5X1)-R6-O-, where R4represents a C1-C5alkylene, R5and R6identical or different, represent a C1-C5alkylene, and X1represents putrescency, spermidine or Perminova the rest.

2. Molecule according to claim 1, in which the oligonucleotide is selected from the group consisting of deoxyribo-, RIBO-, closed (LNA) nucleotides, and their chemical modification or substitution.

3. Molecule according to claim 2, which are modifications or substitutions are phosphorothioate, 2'-fluoro-, 2'-O-alkyl.

4. Molecule according to any one of claims 1 to 3, comprising the marker group, which is a fluorescent agent.

5. Molecule according to any one of claims 1 to 3, in which amino acids are arginine, lysine, ornithine, histidine, diaminopropionic acid.

6. Molecule according to any one of claims 1 to 3, with3'And5'-In sequence.

7. Molecule according to any one of claims 1 to 3, with In-3'And5'sequence.

8. Molecule according to any one of claims 1 to 3, with In-3'And5'Or3'And5'-B3'A5'sequences, and combinations thereof.

9. The production method of oligonucleotide-oligohaline molecules according to any one of claims 1 to 8 by using stepwise synthesis of oligonucleotide synthesizer according phosphoramidites way, including
placement of test tubes containing activated and protected aliocation In, oligonucleotide synthesizer with added to the test tubes oligonucleotides or Vice versa,
- stop synthesis is achieved when the desired length,
- cleavage of the oligomer from the solid media and
- remove the protective groups.

10. The method according to claim 9, in which phosphoramidite reagents selected from the group including
P(OR9)(N(R10)2)-O-R1-(X-R2)n1-X-R3-O-Prot, where R1, R2, R3and index n1 such, as defined by you is e, X represents an appropriately protected NH or NC(NH2)2, R9represents-CH2CH2CN or C1-C5alkyl, R10represents a C1-C5alkyl, or-N(R10)2is a pyrolidine, piperidine or morpholino group, and Prot is a protective group used in oligonucleotide synthesis, such as DMT, MMT;
P(OR9)(N(R10)2)-O-R4-CH(R5X1)-R6-O-Prot, where R4, R5, R6represents a C1-C5alkylene, X1represents an appropriately protected putrescine, spermidine or spermine, R9and R10such as described above.

11. The method according to claim 9 or 10, in which the stepwise synthesis of the oligonucleotide sequence proceeds stepwise synthesis aliocation fragment to produce compounds having the sequence (3'A5'-B).

12. The method according to claim 9 or 10, in which stepwise synthesis aliocation fragment continues sequential synthesis of the oligonucleotide sequence to produce compounds having the sequence (- 3'And5').

13. The method according to claim 9 or 10, comprising the synthesis of mixed sequences.

14. The method according to item 13, including the synthesis of oligonucleotide follower of the awn, keperawanan at both ends (In-3'And5'-In), or katieprice sequences of oligonucleotide sequences [3'And5'In3'And5').

15. The method according to claim 9 or 10, in which activated and protected aliocation get In the protection of amino groups polyamine with subsequent α,ω-bishydroxycoumarin, leading to dilam compatible with oligonucleotide synthesis.

16. As intermediates, phosphoramidite reagents of the formula
P(OR9)(N(R10)2)-O-R1-(X-R2)n1-X-R3-O-Prot, where R1, R2, R3and index n1 such as defined above, X represents an appropriately protected NH or NC(NH2)2, R9represents-CH2CH2CN or lower alkyl, R10represents lower alkyl, or-N(R10)2is a pyrolidine, piperidine or morpholino group, and Prot is a protective group used in oligonucleotide synthesis, such as DMT, MMT;
P(OR9)(N(R10)2)-O-R4-CH(R5X1)-R6-O-Prot, where R4, R5, R6represents a C1-C5alkylene, X1represents an appropriately protected putrescine, spermidine or spermine, R9and R10such as described above.

17. P is the physical alteration of the oligonucleotide-oligohaline molecules according to any one of claims 1 to 8, the method for use in biology and diagnosis, such as PCR, real time PCR, genotyping, in situ hybridization and manufacture of DNA chips.

18. Pharmaceutical compositions containing an effective amount of the oligonucleotide-oligohaline molecules according to any one of claims 1 to 8, in combination with a pharmaceutically acceptable carrier.



 

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FIELD: medicine, pharmaceutics.

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39 cl, 4 tbl

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FIELD: genetic engineering, medicine.

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21 cl, 7 dwg, 3 tbl, 3 ex

FIELD: genetic engineering, medicine.

SUBSTANCE: invention relates to T-cell receptor sequence being detected in patients with extended sclerosis and is useful in diagnosis and therapy. Oligonucleotide including sequence which represents or is derived from 5'-CTAGGGCGGGCGGGACTCACCTAC-3' or nucleotide sequence being fully complementary thereto. Oligonucleotide together with nuclear acid including nearly 15-30 oligonucleotides, which doesn't comprise oligonucleotide sequence and presents in region from Vβ to Jβ of Vβ13.1 gene in T-cell Vβ13.1-subgroup, wherein oligonucleotide and nuclear acid sequences don't present in the same chain of pair sequences of Vβ13.1 gene, is used in Vβ13.1 gene part amplification. In method for detection of LGRAGLTY motive, which is present in T-cell receptors of T-cell Vβ13.1-subgroup, oligonucleotide is used in combination with labeling particle. Once LGRAGLTY motive is detected, development monitoring and treatment are carried out by removing of LGRAGLTY motive-containing peptide.

EFFECT: simplified methods for detection of LGRAGLTY motive in T-cell receptors and treatment of patients with extended sclerosis.

21 cl, 7 dwg, 3 tbl, 3 ex

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex

FIELD: organic chemistry, biochemistry.

SUBSTANCE: invention relates to oligomer comprising at least one nucleoside analogue of L-ribo-CNA of the general formula (Ia) wherein X represents -O-; B represents nitrogen base; P means radical position in an internucleoside linkage followed by monomer or 5'-terminal hydroxy-group; P* means an internucleoside linkage with precede monomer or 3'-terminal hydroxy-group; R2* and R4* mean in common biradical -(CH2)0-1-O-(CH2)1-3-(CH2)0-1-S-(CH2)1-3- or -(CH2)0-1-NR-(CH2)1-3- wherein R means hydrogen atom, alkyl or acyl; R1*, R2, R3*, R5 and R5* mean hydrogen atom. Also, invention proposes nucleoside analogues used in preparing oligomers. Proposed oligomers elicit the enhanced affinity to complementary nucleic acids and can be used as a tool in molecular-biological investigations and as antisense, antigen agents of agents activating genes.

EFFECT: valuable properties of analogues.

15 cl, 3 tbl, 4 dwg, 17 ex

FIELD: molecular biology, medicine, pharmaceutical industry.

SUBSTANCE: method for detecting analyzed DNA sequence involves DNA hybridization with probes and visualization the prepared product wherein probes represent oligonucleotides with length of nucleotide sequence 12-30 nucleotides showing complementary to site of the same size in analyzed DNA that are modified with insertions based on alkyldiols or ethylene glycols. Applying the proposed method provides obtaining more reliable and selective results in detecting analyzed DNA sequences.

EFFECT: improved detecting method of DNA sequence.

11 cl, 12 dwg, 13 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to polynucleotide encoding zwal gene product containing polynucleotide sequence selected from group including a) polynucleotide encoding polupeptide with amino acid sequence with at least 90 % identity to amino acid sequence represented in SEQ ID NO:2; b) polynucleotide which is complementary to polynucleotides from a), as well as primer representing polynucleotide containing at least 15 sequential base pairs of abovementioned polynucleotide.

EFFECT: new zwal gene encoding ionic zwal product.

6 cl, 1 dwg, 1 tbl, 5 ex

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