Site-specific acyl modifier for nucleic acid

Abstract

To provide technology for site-specifically introducing acyl modification at a desired position within a nucleic acid sequence.SOLUTION: The present invention provides a reactive nucleic acid probe that enables acylation of a target nucleic acid within a nucleic acid sequence. The reactive nucleic acid probe is represented by the formula below, N1 and N2 are represented by nucleic acid sequences (including partially modified nucleic acid sequences), X is represented by, for example, the formula below, Y1 is selected from H, OH, and OCH3, and R1 is a linear or branched, saturated or unsaturated alkyl chain.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to site-specific acyl modifiers for nucleic acids. [Background technology]

[0002] Nucleic acid pharmaceuticals such as antisense drugs, siRNA drugs, and mRNA vaccines, as well as nucleic acid reagents used in biochemical experiments, are subjected to various chemical modifications, including natural modifications and artificial modifications, for the purpose of suppressing degradation in the body and within cells, and for the purpose of detecting nucleic acids in the body by labeling. That is, the usefulness of nucleic acids can be improved by chemically modifying them according to the purpose. Therefore, there is a demand for the development of methods for site-specific chemical modification of nucleic acids, and various techniques have been disclosed (Non-Patent Documents 1 to 3).

[0003] Non-Patent Document 1 reports the synthesis of mRNA containing an acetyl modification of cytosine by transcription synthesis using acetyl-modified CTP (ac4CTP) at the 4-amino group. Non-Patent Document 2 reports a method for site-specific chemical synthesis of an oligonucleic acid containing an acetyl-modified cytosine (ac4C) at the 4-amino group.

[0004] Furthermore, Non-Patent Document 3 and Patent Document 1 report a method for selectively introducing a specific chemical modification into a target by using a reactive nucleic acid probe complementary to the target nucleic acid. Although the introduction of specific chemical modifications to target positions in nucleic acids is subject to many technical limitations and is limited to certain structures, Non-Patent Document 3 and Patent Document 1 disclose a method for modifying the target cytosine of a nucleic acid with a nitrosyl group, a 2-vinyliden-1,3-diketo group, or a pyridinyl-keto vinyl group, and a method for modifying the target adenine of a nucleic acid with a pyridinyl-keto vinyl group. In addition, with regard to acetyl group modification, a method for modifying the hydroxyl group at the 2' position of the sugar moiety of a nucleic acid is disclosed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2020-162476 A [Non-patent literature]

[0006] [Non-Patent Document 1] Sinclair, WR, and 9 others, “Profiling Cytidine Acetylation with Specific Affinity and Reactivity”, ACS Chemical Biology, 2017, 12, 2922 - 2926. [Non-Patent Document 2] Bartee, D., et al., “Site-Specific Synthesis of N4 - Acetylcytidine in RNA Reveals Physiological Duplex Stabilization”, Journal of the American Chemical Society, 2022, 144, 3487 - 3496. [Non-Patent Document 3] Sasaki, S., “Development of Novel Functional Molecules Targeting DNA and RNA”, Chemical and Pharmaceutical Bulletin, 2019, 67, 505 - 518. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0007] Prior art acetyl group modification of nucleic acids has problems in that it requires an excess amount of an acetylating agent and is limited to non-specific modification. In addition, the method of Non-Patent Document 2 has a problem that it is not possible to modify already-synthesized or naturally-occurring nucleic acids because it is necessary to incorporate an acetyl modification structure at the stage of nucleic acid synthesis. Furthermore, the method of Non-Patent Document 2 has a problem in that it is based on solid-phase synthesis and therefore has limitations on the synthesis of long-chain nucleic acids. Thus, a technique for site-specifically introducing acyl modifications into a desired position in a nucleic acid sequence has not yet been established.

[0008] In light of the above circumstances, an objective of the present invention is to provide a technique for site-specifically introducing an acyl modification into a desired position in a nucleic acid sequence. [Means for solving the problem]

[0009] As a result of intensive research, the inventors came up with the idea of ​​an acyl group-donor nucleic acid probe that enables acyl group modification of a target nucleic acid base by introducing a reactive acyl group having a specific structure into guanine or thymidine, and completed the invention.

[0010] The present invention comprises the following configurations. [1] A reactive nucleic acid probe capable of modifying a target position in a nucleic acid sequence with an acyl group, The reactive nucleic acid probe is represented by the following formula 1: [ka] N1 and N2 are represented by nucleic acid sequences (including partially modified nucleic acid sequences), X is represented by the following formula 2 or 3: [ka] [ka] Y1 and Y2 are selected from the group consisting of H, OH, and OCH3; R1, R2 are linear or branched, saturated or unsaturated alkyl chains; Reactive nucleic acid probes. [2] The reactive nucleic acid probe according to [1], wherein the alkyl chain is an alkyl chain having 1 to 10 carbon atoms. [3] The reactive nucleic acid probe according to [1], wherein the partial modification in the nucleic acid sequence is 2'-F, 2'-OCH3, or LNA.

[0011] [4] A reactive nucleic acid probe according to any one of [1] to [3], A method for acyl-modifying a nucleic acid, comprising acyl-modifying a nucleic acid sequence complementary thereto or a nucleic acid sequence containing the same. [5] A reactive nucleic acid probe of the following formula 1; [ka] A nucleic acid sequence represented by the following formula 4: Formula 4 is a nucleic acid sequence complementary to Formula 1 or containing the same; [ka] By reacting Chemical 1 with Chemical 4, The method for modifying an acyl group according to [4], wherein the target base X' is modified with an acyl group. [6] A nucleic acid sequence represented by the following formula 5 (including a partially modified nucleic acid sequence): [ka] Z is represented by the following formula 6 or 7: (Y1 and Y2 are selected from H, OH, and OCH3). [ka] [ka] The nucleic acid sequence of the above formula 5 is reacted with a compound represented by the following formula 8, (R1 and R2 are linear or branched, saturated or unsaturated alkyl chains.) [ka] A method for producing a reactive nucleic acid probe according to [1]. Effect of the Invention

[0012] The present invention makes it possible to provide a technique for site-specifically introducing acyl modifications into desired positions in a nucleic acid sequence. [Brief description of the drawings]

[0013] [Figure 1] FIG. 2 shows the results of HPLC analysis of the nucleic acid probe according to Experimental Example I and the reactive nucleic acid probe. [Diagram 2] FIG. 2 shows the results of HPLC analysis of each reaction solution in the acetyl modification reaction according to Experimental Example I. [Diagram 3] FIG. 1 shows a total ion chromatogram (TIC) of the acetyl modification reaction according to Experimental Example I. [Figure 4] FIG. 13 shows the results of HPLC analysis of the nucleic acid probe and the reactive nucleic acid probe according to Experimental Example II. [Diagram 5] FIG. 1 shows a total ion chromatogram (TIC) of the acetyl modification reaction according to Experimental Example II. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will now be described.

[0015] The reactive nucleic acid probe of the present invention is a reactive nucleic acid probe capable of being modified with an acyl group at a desired position in a nucleic acid sequence, and is represented by Chemical Formula 1 below. [ka] In Chemical Formula 1, N1 and N2 are represented as DNA or RNA type nucleic acid sequences. That is, the reactive nucleic acid probe of the present invention can select a nucleic acid sequence according to the nucleic acid sequence to be modified. The combination of reactive nucleic acid probe / nucleic acid sequence to be modified (target nucleic acid) can be DNA type / RNA type, RNA type / DNA type, DNA type / DNA type, RNA type / RNA type, etc. The nucleic acid sequence to be modified does not need to be particularly limited as long as it has a nucleic acid sequence, and various nucleic acids such as artificially synthesized nucleic acid, naturally occurring nucleic acid, long chain nucleic acid, and chemically modified nucleic acid can be modified.

[0016] The nucleic acid sequence may be partially modified for the purpose of improving the stability of the reactive nucleic acid probe itself, etc. Examples of such partial modifications include 2'-OCH3 nucleic acid, 2'-Fluoro nucleic acid, and LNA.

[0017] In Chemical formula 1, X is represented by Chemical formula 2 or 3 below. [ka] [ka] Chemical formula 2 is a guanine-type reactive nucleic acid probe, which enables acyl group modification of the paired nucleic acid base as shown in Chemical formula 9 below. [ka] Taking cytosine as an example, a reactive nucleic acid probe of the guanine type has a cytosine N4 position as shown in the following formula 10. Or, as shown in Chemical formula 11, Similarly, the acyl group is modified at the N6 position for adenine, the N2 position for guanine, the N2 position for uracil, Alternatively, the sugar hydroxyl groups of these bases Acyl group modification is performed. [ka] [ka] Chemical formula 3 is a thymine-type reactive nucleic acid probe, which enables acyl group modification of the paired nucleic acid base as shown in Chemical formula 12 below. [ka] Taking adenine as an example, a thymine-type reactive nucleic acid probe is shown below as formula 13, which is adenine N6 position. Or, as shown in Chemical formula 14, Similarly, acyl modification is performed at the N4 position for cytosine, the N2 position for guanine, the N2 position for uracil, Alternatively, the sugar hydroxyl groups of these bases Acyl group modification is performed. [ka] [ka]

[0018] The acyl modification reaction of the target base can be carried out, for example, by mixing the target nucleic acid with a reactive nucleic acid probe in a buffer solution under neutral or alkaline conditions. The reaction temperature for the above modification reaction can typically be from 15°C to 40°C, and preferably 15°C or 37°C. The reaction time in a buffer solution under alkaline conditions is typically 30 minutes to 3 hours, preferably 1 hour, and in a buffer solution under neutral conditions is typically 18 to 48 hours, preferably 24 hours.

[0019] In Chemical Formula 2 or 3, Y1 and Y2 are selected from H, OH, and OCH3. That is, X itself can be appropriately selected as a DNA type (H), an RNA type (OH), or a degradation-inhibited type (OCH3).

[0020] In Formula 2 or Formula 3, R1 and R2 are represented as linear or branched, saturated or unsaturated alkyl chains. For the alkyl chain, the number of carbon atoms can be adjusted taking into consideration the steric hindrance and size of the reactive nucleic acid probe itself, and can typically be 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 4, and most preferably 1 to 3. Furthermore, an unsaturated alkyl chain can be used as a functional group for a click reaction, for example, by making the terminal an alkyne.

[0021] The reactive nucleic acid probe of the present invention can perform acyl group modification on a nucleic acid sequence complementary thereto, and can also perform acyl group modification on a nucleic acid sequence containing a nucleic acid sequence complementary thereto. In other words, since acyl group modification is possible as long as the double strand of the nucleic acid probe and the target nucleic acid is stable in a buffer solution, there is no need to limit the chain length of the reactive nucleic acid probe, and the same applies to the corresponding target nucleic acid.

[0022] The method for modifying the reactive nucleic acid probe of the present invention is carried out by reacting the reactive nucleic acid probe of Chemical formula 1 with the nucleic acid sequence of Chemical formula 4. N1, N2, N'1, N'2 are represented as nucleic acid sequences of DNA or RNA type. [ka] [ka] The nucleic acid sequence represented by Chemical formula 4 is a nucleic acid sequence complementary to Chemical formula 1, or a nucleic acid sequence containing the same. By reacting Chemical formula 1 with Chemical formula 4, X' can be modified with an acyl group. That is, in Chemical formula 1 and Chemical formula 4, if N1 and N'1, and N2 and N'2 are complementary, then Chemical formula 1 and Chemical formula 4 are complementary and acyl group modification to X' is possible. Note that N1 is the 5' side of the nucleic acid and N'1 is the 3' side of the nucleic acid. In addition, when Chemical 4 is a longer base sequence than Chemical 1 and Chemical 4 contains a sequence complementary to Chemical 1, X' can be modified with an acyl group.

[0023] The reactive nucleic acid probe of the present invention can be produced by reacting Chemical Formula 5 with Chemical Formula 8. [ka] [ka] (In the formula 8, R is the same as R1 or R2 and is represented as a linear or branched, saturated or unsaturated alkyl chain.)

[0024] The formula 5 is represented as a nucleic acid sequence (including partially modified nucleic acid sequences). In the formula 5, Z is represented by formula 6 or formula 7, and Y1 and Y2 are selected from H, OH, and OCH3. [ka] [ka]

[0025] As an example of the case where Y1 in Chemical formula 6 is H, an oligonucleotide containing 6-thio-2'-deoxyguanosine is synthesized by a known method, and then reacted with a (Bromomethyl)acyl sulfide reagent (Chemical formula 8, R = R1) having the substituent (R) to be introduced in a buffer solution. [ka]

[0026] As an example of the case where Y1 is H in Chemical formula 7, an oligonucleotide containing 4-thio-2'-deoxythymidine is synthesized by a known method, and then reacted with a (Bromomethyl)acyl sulfide reagent (Chemical formula 8, R = R2) having a substituent (R) to be introduced in a buffer solution as shown in Chemical formula 12 below. [ka] EXAMPLES

[0027] The present invention will now be described in detail with reference to examples.

[0028] <<Experimental Example I. Acetyl modification of RNA using a guanine-type reactive nucleic acid probe>> A 1.15-mer RNA was targeted, and acetyl modification (a*, g*, c*, or u*) was performed on the seventh base (a, g, c, or u) from the 5'-end. 2. The nucleic acid probe is a 15-mer DNA that has 6-thio-2'-deoxyguanosine (X) at the complementary position of the target base (a, g, c, or u). The reactive nucleic acid probe is synthesized from the nucleic acid probe and has 6-(thiomethyl)acetyl sulfide-2'-deoxyguanosine (X'). 3. The nucleic acid sequences are shown in Table 1. [Table 1]

[0029] 4. Synthesis of reactive nucleic acid probe (guanine type) (1) A nucleic acid probe containing 6-thio-2'-deoxyguanosine (50 μM) and (Bromomethyl)acetyl sulfide (50 mM) in CAPS buffer (250 mM, pH 10) were prepared at 0°C and reacted at the same temperature for 30 minutes to synthesize a reactive nucleic acid probe. (2) Analysis of the reaction and purification of the reactive nucleic acid probe were performed by HPLC (JASCO). The HPLC analysis results are shown in Figure 1. (3) A peak of the reactive nucleic acid probe was obtained at a position with a later retention time than the nucleic acid probe. This was separated and purified by HPLC and used for the subsequent acetylation of RNA.

[0030] 5. Reaction example of acetyl modification of RNA (1) The reactive nucleic acid probe (7.5 μM) and the target nucleic acid (5 μM) were mixed in a phosphate buffer (50 mM, pH 8.5) containing NaCl (100 mM) and incubated at 37°C for 22 hours to obtain acetyl-modified RNA. (2) Reaction analysis was performed by HPLC (JASCO). The HPLC results are shown in Figure 2. (3) A peak of acetyl-modified nucleic acid was obtained at a position with a later retention time compared to the target nucleic acid. (4) Mass spectrometry identified the following: acetyl-modified nucleic acid (adenine type) (calcd. 4823, found 4823), acetyl-modified nucleic acid (guanine type) (calcd. 4839, found 4839), acetyl-modified nucleic acid (cytosine type) (calcd. 4799, found 4799), acetyl-modified nucleic acid (uracil type) (calcd. 4800, found 4800). (5) The cytosine-type acetylation reaction was analyzed by UPLC / MS (waters). The results of the UPLC / MS total ion chromatogram (TIC) are shown in Figure 3. (6) Mass spectrometry identified the peaks of the target nucleic acid (cytosine type) (calcd. 4757, found 4757), acetyl-modified nucleic acid (cytosine type) (calcd. 4799, found 4799), nucleic acid probe (guanine type) (calcd. 4631, found 4631), and reactive nucleic acid probe (guanine type) (calcd. 4719, found 4719). 6. These results show that the acetylation of any base, adenine, guanine, cytosine, or uracil, is possible using the reactive nucleic acid probe (guanine type).

[0031] <<Experimental Example II: Acetyl modification of RNA using a thymine-type reactive nucleic acid probe>> A 1.15-mer RNA was targeted, and the 9th adenine from the 5'-end was modified with acetyl (a*). 2. The nucleic acid probe is a 15-mer DNA that has 4-thio-2'-deoxythymidine (Y) at the complementary position of the target adenine. The reactive nucleic acid probe is synthesized from the nucleic acid probe and has 4-(thiomethyl)acetyl sulfide-2'-deoxythymidine (Y'). 3. The nucleic acid sequences are shown in Table 2. [Table 2]

[0032] 4. Synthesis of reactive nucleic acid probe (thymine type) (1) A nucleic acid probe containing 4-thio-2'-deoxythymidine (50 μM) and (Bromomethyl)acetyl sulfide (50 mM) in CAPS buffer (250 mM, pH 10) were prepared at 0°C and reacted at the same temperature for 30 minutes to synthesize a reactive nucleic acid probe. (2) Analysis of the reaction and purification of the reactive nucleic acid probe were performed by HPLC (JASCO). The HPLC analysis results are shown in Figure 4. (3) A peak of the reactive nucleic acid probe was obtained at a position with a later retention time than the nucleic acid probe. This was separated and purified by HPLC and used for the subsequent acetylation of adenine.

[0033] 5. Reaction example of acetyl modification of adenine (1) The reactive nucleic acid probe (7.5 μM) and the target nucleic acid (5 μM) were mixed in a carbonate buffer (50 mM, pH 10) containing NaCl (100 mM) and incubated at 15 °C for 46 hours. This resulted in the production of acetyl-modified RNA. (2) Reaction analysis was performed by UPLC / MS (waters). The results of the UPLC / MS TIC analysis are shown in Figure 5. (3) Peaks of acetyl-modified nucleic acids were obtained at a position with a later retention time compared to the target nucleic acid. Mass spectrometry identified the peaks of the target nucleic acid (calcd. 4758, found 4758), acetyl-modified nucleic acid (calcd. 4800, found 4800), nucleic acid probe (calcd. 4615, found 4615), and reactive nucleic acid probe (calcd. 4703, found 4703).

[0034] 6. Both Experimental Example I and Experimental Example II were analyzed under the following conditions. [HPLC analysis conditions] Osaka Soda CAPCELL PAK C18 MG (4.6×250mm) A: 0.1M TEAA buffer pH7.0 + 5% ACN, B: ACN, B: 0~30% / 20 min, UV: 254nm, Flow rate: 1.0 mL / min, Column oven: 40℃ [UPLC / MS analysis conditions] ACQUITY UPLC: Oligonucleotide BEH C18 Column, 130Å, 1.7μm, 2.1x50 mm, A: 15 mM TEA, 400 mM HFIP pH7.9, B: A / MeOH=1 / 1, B:35~70% / 10min, UV: 254nm, flow rate: 0.2mL / min, column oven: 40℃, MS system: Xevo G2-XS Qtof, Mode: ESI negative resolution, continuum, Cone voltage: 40V, Capillary voltage: 4kV, desolvation: 600℃, desolvation gas flow: 700L / hr [Industrial Applicability]

[0035] The present invention is useful for site-specific acyl modification of nucleic acid pharmaceuticals and research nucleic acid reagents.

Claims

1. A reactive nucleic acid probe capable of modifying a nucleic acid sequence with an acyl group at a desired position, The reactive nucleic acid probe is represented by the following formula 1: 【Chemistry 1】 N 1 , N 2 is represented by a nucleic acid sequence (including a partially modified nucleic acid sequence), X is represented by the following formula 2 or 3: 【Chemistry 2】 【Chemistry 3】 Y 1 , Y 2 are H, OH, and OCH. 3 is selected from either R 1 , R 2 is a linear or branched, saturated or unsaturated alkyl chain; Reactive nucleic acid probes.

2. 2. The reactive nucleic acid probe according to claim 1, wherein the alkyl chain is an alkyl chain having 1 to 10 carbon atoms.

3. Partial modifications in the nucleic acid sequence include 2'-F, 2'-OCH 3 2. The reactive nucleic acid probe according to claim 1, which is LNA.

4. A reactive nucleic acid probe according to any one of claims 1 to 3, A method for acyl-modifying a nucleic acid, comprising acyl-modifying a nucleic acid sequence complementary thereto or a nucleic acid sequence containing the same.

5. A reactive nucleic acid probe of the following formula 1, 【Chemistry 1】 A nucleic acid sequence represented by the following formula 4: Formula 4 is a nucleic acid sequence complementary to Formula 1 or containing the same; 【Chemistry 4】 By reacting Chemical 1 with Chemical 4, The method for modifying an acyl group according to claim 4, wherein X' is modified with an acyl group.

6. A nucleic acid sequence represented by the following formula 5 (including a partially modified nucleic acid sequence): 【Chemistry 5】 Z is represented by the following formula 6 or 7: (Y 1 , Y 2 are H, OH, and OCH. 3 (chosen from either 【Chemistry 6】 【Chemistry 7】 The nucleic acid sequence of the above formula 5 is reacted with a compound represented by the following formula 8, (R 1 , R 2 is a linear or branched, saturated or unsaturated alkyl chain; 【Chemistry 8】 A method for producing the reactive nucleic acid probe according to claim 1 .

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