RU2305108C1 - OLIGONUCLEOTIDE-PEPTIDE CONJUGATE WITH ABILITY TO CLEAVE RNA PHOSPHODIESTER BONDS IN 5'GpN3' SEQUENCES - Google Patents

Abstract

FIELD: bioorganic chemistry.

SUBSTANCE: disclosed is oligonucleotide-peptide conjugate of general formula 3'TTCTCTAGG5'-dRib-dRib-dRib-N-terminal-Leu-Arg-Leu-Arg-Leu-Arg-Arg-Gly-NH₂-C-terminal (wherein T is thymine residue; C is cytosine residue; A is adenosine residue; G is guanosine residue; drib is deoxyribose residue; Leu is leucine residue; Arg is arginine residue; Gly is glycine residue). Said conjugate cleaves phosphodiester bonds in single-stranded RNA sites in 5'GpN3' (wherein G is guanosine; N is any ribonucleotide) sequences only.

EFFECT: new conjugate being functional analog of T1 ribonuclease.

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Description

The invention relates to the field of bioorganic chemistry and relates to the production of an oligonucleotide peptide conjugate having the ability to cleave phosphodiester bonds in single-stranded sections of RNA in ^{5 '} GpN ^3' sequences, which can be used to study the structure of RNA, as well as RNA-protein, RNA-DNA complexes.

The directed cleavage of ribonucleic acid (RNA) into given sequences is an urgent problem due to the need to manipulate RNA molecules in molecular biological studies and to create therapeutic drugs acting on RNA of infectious agents and mRNA encoding proteins responsible for the development of pathological conditions.

It is known to use the enzyme RNase T1 from Aspergillus oryzae for specific cleavage of phosphodiester bonds in single-stranded regions of RNA in ^{5 '} GpN ^3' sequences [1]. The disadvantage of this enzyme is its low stability at room temperature and the ability to unfold an RNA molecule, which inevitably distorts the experimental results. In addition, RNase T1 operates in a narrow range of conditions.

One approach to the creation of synthetic compounds that cleave specific RNAs is the creation of conjugates of oligonucleotides with catalytically active groups that can cause the cleavage of phosphodiester RNA bonds. Several types of conjugates based on oligodeoxyribonucleotides carrying groups capable of catalyzing RNA cleavage are known: imidazole residues [2, 3], imidazole with an attached amino group [4], bis-imidazole constructs [5], ethylenediamine or propylene diamine [6], as well as derivatives terpyridine, chelating copper ions [7], and various complexes of lanthanides [8]. In this case, oligonucleotides fulfill the task of delivering catalytically active groups to a selected site of the target molecule.

A common disadvantage of the known conjugates is the low efficiency of RNA cleavage, in addition, these conjugates cleave RNA at the most labile bonds — CPA and UpA. Another specificity of cleavage with RNA conjugates has not been demonstrated.

Closest to the claimed prototype is an oligonucleotide peptide conjugate, which is an oligonucleotide covalently attached to a peptide of the general formula: ACAAACC-p-Leu-Arg-Leu-Arg-Leu-Arg-Leu-Arg-Gly-NH ₂ , where C is cytosine residue, A - adenosine residue, Leu - leucine residue, Arg - arginine residue, Gly - glycine residue [9]. This oligonucleotide peptide conjugate cleaves the phosphodiester bonds of RNA in the sequences of CPA, UpA and GpN. The oligonucleotide in the composition of this type of conjugate is not complementary to the target RNA; it acts as an adapter for the catalytically active peptide conformation and modulates the specificity of cleavage [9].

The disadvantage of this conjugate is the mixed specificity of RNA cleavage - according to the sequences of the two types Pyr-pA (where Pyr is the pyrimidine nucleotide, A is adenosine) and GpN (G is guanosine, N is any nucleotide).

An object of the present invention is to provide an oligonucleotide peptide conjugate that cleaves RNA phosphodiester bonds with high efficiency exclusively in ^{5 ′} GpN ^{3 ′} sequences.

This goal is achieved by the proposed oligonucleotide peptide conjugate having the ability to cleave phosphodiester RNA bonds in ^{5 '} GpN ^3' sequences.

The inventive oligonucleotide peptide conjugate consists of an oligonucleotide moiety ( ^{5 '} GGATCTCTT ^3' ), a peptide moiety (Leu-Arg-Leu-Arg-Leu-Arg-Leu-Arg-Gly-NH ₂ ) and the deoxyribose phosphate linker d-dibR ) and has the following general formula:

^{3 '} TTCTCTAGG ^5' -dRib-dRib-dRib- ^N-terminus Leu-Arg-Leu-Arg-Leu-Arg-Leu-Arg-Gly-NH ₂ ^C-terminus , where: T - thymine residue, C - cytosine residue , A is the adenosine residue, G is the guanosine residue, dRib is the deoxyribose residue, Leu is the leucine residue, Arg is the arginine residue, Gly is the glycine residue.

The oligonucleotide peptide conjugate (code name re-9) has a molecular weight of 4.4 kDa.

The RNA cleavage reaction is carried out under the following conditions: buffer, 50 mM Tris-HCl, pH 3.8-9.0, 2.5 · 10 ^-4 M RNA carrier, 0.1-1 mM EDTA, 0-200 mM KCl, 0-5 mM MgCl ₂ . Labeled ³² P at the 5'-end of RNA, the concentration of which did not exceed 5 · 10 ^-7 M, was incubated at 30-40 ° C for 3-24 hours in the presence of 1-100 μM oligonucleotide peptide conjugate repr-9. The cleavage products are analyzed by electrophoresis in 12-18% PAAG under denaturing conditions. To identify cleavable phosphodiester bonds, the preparations of the corresponding RNA hydrolyzed with 2 M imidazole, pH 7.0, and RNase T1 under denaturing conditions are separated in parallel on the same gel.

The invention is illustrated by the following examples of specific performance.

Example 1. Synthesis of oligonucleotide peptide conjugate re-9

The conjugate was prepared according to the method described previously [10]. Oligodeoxyribonucleotide ^{5 '} GGATCTCTT ^{3' was} synthesized by the solid-state automatic phosphitamide method on an automatic synthesizer. The peptide (LR) ₄ G-NH _{2 was} attached to the oligodeoxyribonucleotide by forming a phosphamide bond between the 5'-terminal phosphate group of the linker consisting of deoxyribose residues and the α-amino group of the N-terminal leucine residue. Isolation of the oligonucleotide peptide conjugate pop-9 after synthesis was carried out using reverse phase HPLC on a column with LiChrosorb RP-18 sorbent. The homogeneity of the oligonucleotide peptide conjugate pop-9 was tested by electrophoresis in 20% denaturing PAAG followed by staining of the nucleotide material with Stains-all.

Example 2

The oligonucleotide peptide conjugate repr-9 was used to study the structure of RNA 21 and riboduplex based on it.

The cleavage of RNA 21 and the riboduplex based on it with the oligonucleotide peptide conjugate repr-9 was carried out in 50 mM Tris-HCl buffer, pH 7.0, containing 200 mM KCl, 1 mM EDTA. Labeled ³² P at the 5'-end of RNA 21 and a riboduplex based on it at a concentration of 10 ^-7 M were incubated in the presence of 10 μM oligonucleotide peptide conjugate repr-9 at 37 ° C for 5-24 hours.

Figure 1 shows the structure of RNA 21 and riboduplex based on it (A) and an analysis of the cleavage of RNA 21 and riboduplex based on it oligonucleotide peptide conjugate re-9 (B) in 12% denaturing PAAG. Lane — RNA cleavage with 2 M imidazole, pH 7.0, under denaturing conditions; lane T1 - cleavage of RNase T1 at a concentration of 10 units Act. under denaturing conditions; lanes C — incubation of RNA 21 and riboduplex based on it in the absence of a conjugate for 8 hours. RNA 21 and riboduplex based on it were incubated under standard conditions in the presence of 10 μM conjugate at 37 ° C for 1-24 hours. The incubation time is indicated above .

In RNA 21, under the action of the oligonucleotide peptide conjugate of re-9, 5 bonds are cleaved: G ₁₀ , G ₁₁ , G ₁₅ , G ₁₆ and G ₁₉ . With an increase in incubation time, the cleavage pattern remains constant and no other cleavage sites appear. The oligonucleotide peptide conjugate of re-9, even with prolonged incubation with riboduplex, does not cause its cleavage. The data obtained allow us to conclude that the oligonucleotide peptide conjugate of pp-9 is an artificial ribonuclease capable of cleaving phosphodiester bonds in single-stranded sections of RNA in ^{5 '} GpN ^3' sequences.

Example 3

The oligonucleotide peptide conjugate of re-9 was used to study the structure of RNA 96 in solution.

Figure 2 shows the structure of RNA 96 (A) and an analysis of the cleavage of RNA 96 by the oligonucleotide peptide conjugate of re-9 (B) in 12% denaturing PAAG.

Lanes L and T1 — RNA 2 M cleavage with imidazole, pH 7.0, and RNase T1 under denaturing conditions, respectively; lane C - incubation of RNA in the absence of the receptor-9 oligonucleotide peptide conjugate under standard conditions for 8 hours. Labeled ³² P at the 5'end of RNA 96 were incubated in 50 mM Tris-HCl buffer, pH 7.0, containing 1 mM EDTA, in the presence of 10 μM conjugate at 37 ° C for 3-8 hours. The incubation time is indicated above. The cleavage sites of RNase T1 and the oligonucleotide peptide conjugate repr-9 are indicated on the left, the sites of spontaneous hydrolysis are indicated on the right, respectively. Figure 2 shows that the RNA cleavage sites of the oligonucleotide peptide conjugate of pp-9 and RNase T1 are the same.

Example 4

Comparison of the properties of the oligonucleotide peptide conjugate of re-9 and RNase T1.

Figure 3 presents the analysis of the cleavage products of ³² P-labeled at the 5'-end of RNA 96 with the oligonucleotide peptide conjugate repr-9 and RNase T1 in a 12% polyacrylamide denaturing gel. Lane — RNA cleavage with 2 M imidazole, pH 7.0, under denaturing conditions; lane C - incubation of RNA in the absence of the oligonucleotide peptide conjugate peer-9 and RNase T1 under appropriate conditions for 8 hours

Lanes d: RNA was incubated in buffer d in the presence of RNase T1 at a concentration of 3 and 5 units. Act. at 50 ° C for 15 minutes;

lane d1: RNA was incubated in buffer d1 in the presence of 10 μM oligonucleotide peptide conjugate repr-9 at 37 ° C for 5 and 8 hours; lanes d2, s / d, n: RNA was incubated in d2, s / d and n buffers at 37 ° С in the presence of the oligonucleotide peptide conjugate pop-9 (10 μM) for 5 and 8 h and RNase T1 (3, 5 units. Act.). The incubation time with the oligonucleotide peptide conjugate repr-9 is indicated above. The hydrolysis sites of RNase T1 and the oligonucleotide peptide conjugate repr-9 are indicated on the left, the sites of RNA cleavage in the controls are indicated on the right. Figure 3 shows that the inventive oligonucleotide peptide conjugate pop-9 is a functional analogue of ribonuclease T1 and can be used as a reagent to determine the location of the sequences of phosphodiester bonds ^{5 '} GpN ^3' in single-stranded sections of RNA.

Information sources

1. Takahashi, K. and Moore, S. Part B, Ed. Boyer, P. D., Academic Press, New York, 435 (1982) In: The Enzymes: XV.

2. J. Hovinen, A. Guzaev, A. Azhayev and H. Lönneberg, J. Org. Chem., 1995, 60, 2005.

3. M.A. Reynolds, T. A. Beck and L. J. Arnold, Nucleic Acids Res., 1996, 24, 760.

4. K. Ushijima and H. Takaku, Biochim. Biophys. Acta, 1998, 1379, 217.

5. V. Silnikov, G. Zuber, J.-P. Behr, R. Gige. and V.Vlassov. Phosphorus, Sulfur, Silicon Relat. Elem., 1996, 109-110, 277.

6. M. Komiyama and T. Inokawa, J. Biochemistry, 1994, 116, 719.

7. J.K. Bashkin, E. I. Frolova and U.S. Sampath. J. Am. Chem. Soc., 1994, 116, 5981.

8. V.M. Belkov, H.F. Krynetskaya, E.M. Volkov, Z.A.Shabarova, H.Yu. Kraynova, G.N. Novodarova, M.E. Volpin. Bioorganic chemistry, 1995, 21, 446 [Russ. J. Bioorg. Chem., 1995, 21 (Engl. Transl)].

9. Mironova N.L., Pyshnyi D.V., Ivanova E.M., Zenkova M.A., Gross H.J., Vlassov V.V. Covalently attached oligodeoxyribonucleotides induce RNase activity of a short peptide and modulate its base specificity. Nucleic Acids Res. 2004 Mar 26; 32 (6): 1928-36.

10. Zarytova V.F., Ivanova E.M., Yarmolyuk S.N., Alekseeva I.V. Synthesis of oligonucleotidyl- (5'-N) containing arginine. Biopolymers and cell. 1988.V. 1. S.220-222.

Claims (1)

An oligonucleotide peptide conjugate having the ability to cleave RNA phosphodiester bonds in ^{5 ′} GpN ^{3 ′} sequences, having the following general formula: ^{3 '} TTCTCTAGG9-dRib-dRib-dRib- ^N-terminus Leu-Arg-Leu-Arg-Leu-Arg-Leu-Arg-Gly-NH ₂ ^C-terminus , where T is the thymine residue, C is the cytosine residue, A is the adenosine residue, G is the guanosine residue, dRib is the deoxyribose residue, Leu is the leucine residue, Arg is the arginine residue, Gly is the glycine residue.

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