WO1999000401A1

WO1999000401A1 - 3'-monophosphorylating oligonucleotides

Info

Publication number: WO1999000401A1
Application number: PCT/CN1998/000102
Authority: WO
Inventors: Chang-De Lu; Min Zhuang; Yu-Lei Xia
Original assignee: Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences
Priority date: 1997-06-28
Filing date: 1998-06-26
Publication date: 1999-01-07
Also published as: CN1203919A; CN1261893A; JP2002508768A; CN1060177C; AU7905198A

Abstract

3'-Monophosphorylating oligonucleotides are provided, which stability in serum and endocellular is markedly higher than that of non-modified oligonucleotides and a little higher than that of phosphorothioate-modified oligonucleotides demonstrated by studying them in vitro and cell, which ability to uptake by cell is higher than that of non-modified oligonucleotides and phosphorothioate-modified oligonucleotides. Its products of metabolic degradation do not have any toxycity and side effect, due to 3'-monophosphorylating oligonucleotides not containing non-natural modification elements. Therefore, when used as therapeutic agents, 3'-monophosphorylating oligonucleotides are more advantageous and safer than phosphorothioate-modified oligonucleotides.

Description

3，-单磷醴化寡核苷醴技术领域 3, -monophosphorylated oligonucleoside hydrazone Technical field

本发明涉及修饰的寡核苷酸，尤指 3'-0H单磷酸化修饰的寡核苷酸，以增加稳定性，降低毒付作用，该单磷酸化修饰的寡核苷酸可用作治疗的药物。技术背景 The present invention relates to modified oligonucleotides, especially 3'-0H monophosphorylated modified oligonucleotides to increase stability and reduce toxic side effects. The monophosphorylated modified oligonucleotides can be used as a treatment Drug. technical background

寡核苷酸包括反义寡核苷酸、抗基因寡核苷酸 (亦称三螺旋形成寡核苷酸) 等可用于抑制基因的表达，是近年来发展起来的一种新的治疗方法（Wagner, RW, Nature, 1994， 372， 333-335; Crook, ST, Annu. Rev. Pharmacol. Toxica l. , 1992， 32, 329-376; Helene, C， Eur. J. Cancer, 1994， 30A, 1721-1726 ; Crooke, ST, Ant i sense Nucle ic ac ids Drug Dev. , 1996， 6， 141-147)。其作用机制包括阻遏病毒基因的复制、转录和翻译，也可阻遏人体内有害基因的转录和翻译，以及通过体内的 RNase H酶的作用使靶 RNA降解，是一类专一性高、毒副作用小的治疗药物。目前已进入临床试验的反义寡核苷酸有抗 HIV的 GEM91 ( II期），抗炎症的 ISIS2302 ( II期），抗癌症的 ISIS3521 ( I期）及 ISIS5132 ( I期），抗艾滋病患者的 CMV视网膜炎的 ISIS2922 ( III 期） , 治疗慢性髓性白血病的 LR - 3001 ( I期）等 ( Genet ic Engineer ing News, 1996, 16， 29-34)。 Oligonucleotides include antisense oligonucleotides, anti-gene oligonucleotides (also known as triple helix-forming oligonucleotides), etc., which can be used to suppress gene expression, and are a new therapeutic method developed in recent years Wagner, RW, Nature, 1994, 372, 333-335; Crook, ST, Annu. Rev. Pharmacol. Toxica l., 1992, 32, 329-376; Helene, C, Eur. J. Cancer, 1994, 30A, 1721-1726; Crooke, ST, Ant i sense Nucle ic ac ids Drug Dev., 1996, 6, 141-147). Its mechanism of action includes blocking the replication, transcription, and translation of viral genes, as well as the transcription and translation of harmful genes in the human body, and the degradation of target RNA by the action of RNase H enzymes in the body. It is a class of highly specific and toxic side effects Small therapeutic drugs. Antisense oligonucleotides currently in clinical trials include anti-HIV GEM91 (Phase II), anti-inflammatory ISIS2302 (Phase II), anti-cancer ISIS3521 (Phase I) and ISIS5132 (Phase I), anti-AIDS patients CMV retinitis ISIS2922 (Phase III), LR-3001 (Phase I) for the treatment of chronic myelogenous leukemia (Genet ic Engineer ing News, 1996, 16, 29-34).

由于寡核苷酸片段进入人体后，易受到人体内酶***的作用而被降解，即进入人体的寡核苷酸未达到靶器官、靶细胞就被降解因而不能达到预期的治疗效果（Hoke, G. D.， et a l. Nuc le ic Ac ids Res. , 1991, 19， 5734-5748) ₀ 因此，提高寡核苷酸的稳定性，尤其是提高其抗酶解能力显得非常关键，这样, 不仅可以提高疗效、减少药品用量，亦可进一步降低治疗成本和减少副作用。 Since the oligonucleotide fragments enter the human body, they are susceptible to degradation by the enzyme system in the human body, that is, the oligonucleotides entering the human body do not reach the target organ, and the target cells are degraded, so the expected therapeutic effect cannot be achieved (Hoke, GD, et a l. Nuc le ic Ac ids Res., 1991, 19, 5734-5748) ₀ Therefore, it is very important to improve the stability of the oligonucleotide, especially its anti-enzymatic ability. In this way, not only can Increasing the efficacy and reducing the amount of medicine can also further reduce the cost of treatment and reduce side effects.

为了提高寡核苷酸的稳定性，延长其在人体内的半衰期，人们采用不同的方法对寡核苷酸进行了一系列的化学修饰和结构改造，已有许多文献报道，例如：釆用硫磷酸二酯键代替磷酸二酯键的方法（W0 9115500, 1991) ; 将两个寡核苷酸片段以 3'与 3'相连，或 5'与 5'相连（EP 464638 , 1992)等。目前，反义寡核苷酸大多釆用化学修饰方法来增加其稳定性，在诸多化学修饰中，硫磷酸修饰被认为是最理想的一种。但是，它必竟是非天然的，进入细胞后异质性大。而且其合成价格也比非修饰的寡核苷酸高，其化学稳定性也不如天然的磷酸二酯键。硫磷酸修饰寡核苷酸还具有非专一性作用，它能与细胞内的一些重要的蛋白质相结合，影响细胞的信号传递及其他生物功能；它具有很强的免疫原性，可以刺激机体的免疫***；另一方面，釆用硫磷酸二酯键替代物，在体内的代谢降解产物亦可能对人体产生毒副作用（Stein CA, Trends in Biotechnology, 1996, 14, 147-149)。为了寻找更好的修饰方法，本发明进行了进一步的研究。发明目的 In order to improve the stability of the oligonucleotide and extend its half-life in the human body, people use different A series of chemical modifications and structural modifications have been carried out on the oligonucleotides, which have been reported in many literatures, for example: 釆 The method of replacing phosphodiester bonds with thiophosphodiester bonds (W0 9115500, 1991); Oligonucleotide fragments are connected 3 'to 3' or 5 'to 5' (EP 464638, 1992). At present, most of the antisense oligonucleotides are chemically modified to increase their stability. Among many chemical modifications, phosphorothioate modification is considered to be the most ideal one. However, it must be unnatural and has a large heterogeneity after entering the cell. Moreover, its synthetic price is higher than that of unmodified oligonucleotides, and its chemical stability is not as good as that of natural phosphodiester bonds. Thiophosphate-modified oligonucleotides also have a non-specific effect. It can combine with some important proteins in the cell and affect the cell's signal transmission and other biological functions. It has strong immunogenicity and can stimulate the body. On the other hand, the substitution of thiophosphodiester bonds with metabolic replacement products in the body may also have toxic and side effects on the human body (Stein CA, Trends in Biotechnology, 1996, 14, 147-149). In order to find a better modification method, the present invention conducts further research. Object of the invention

本发明的目的是提供一种 3' -单磷酸化寡核苷酸，以增加寡核苷酸稳定性，降低毒副作用。发明内容 The object of the present invention is to provide a 3'-monophosphorylated oligonucleotide to increase the stability of the oligonucleotide and reduce the toxic and side effects. Summary of the Invention

本发明提供一种 3' -单磷酸化寡核苷酸，其结构式可用 5' d (NNN……誦) ρ3'或 ol igo (dN) - 3' P表示，式中 N=A、 G、 C、 T; ρ3'或 y p = ₃'为单磷酸基团。它是釆用 3' -磷酸固相柱（ 3' - phosphate CPG Glen Research公司产品，全称 2_ [ 2- ( 4, 4，-二甲氧基三苯甲氧基）乙磺酰 ] 乙基-丁二酰长链烷基胺-微孔玻璃珠（2- [ 2- ( 4, 4， - Dimethoxy tri tyloxy ) ethylsulfomyl ] ethyl - succinoyl long chain alkylamino-CPG) ) , 在 ABI 391EP DNA合成仪上合成，经浓氨水脱去保护基得 3' -单磷酸化寡核苷酸。为了显示本发明 3 -单磷酸化寡核苷酸对酶降解的稳定性，用蛇毒磷酸二酯酶代表体内的 3'—5'核酸外切酶，用 DNase I代表体内的内切核酸酶在体外比较了不修饰的寡核苷酸、 3' -单磷酸化寡核苷酸、硫磷酸修饰的寡核苷酸及 y 部分硫磷酸修饰的寡核苷酸对 3'→ 5 '核酸外切酶和内切核酸酶的稳定性，进一步还比较了它们在血清中及细胞内的稳定性。实验证明经 3' -磷酸化修饰的寡核苷酸片段具有抗蛇毒磷酸二酯酶的性质，在血清中和细胞内的稳定性明显高于 3'不修饰的或 y部分硫磷酸修饰的寡核苷酸，略高于硫磷酸修饰的寡核苷酸。而且， 3' -磷酸化修饰不影响寡核苷酸进入细胞的速度，这一点优于硫磷酸修饰的寡核苷酸。上述结果说明血清和细胞中主要存在的是 3'→5 '外切核酸酶，而该酶作用时需提供具有 3' - 0H的底物，当寡核苷酸 3' 0H基团被磷酸化后，就不能作为 3'→5'外切核酸酶的底物，因而能抗 3 →5'外切核酸酶作用，延长了在人体血清中的滞留时间，能够更有效地被运输到靶细胞中。在细胞中由于 3' -单磷酸化寡核苷酸稳定性高，因此能更有效地与目的基因的互补序列相结合。 The present invention provides a 3'-monophosphorylated oligonucleotide, whose structural formula can be expressed by 5 'd (NNN ...) ρ3' or ol igo (dN)-3 'P, where N = A, G, C, T; ρ3 'or yp = ₃ ' is a monophosphate group. It is a 3'-phosphate solid phase column (3'-phosphate CPG Glen Research company product, full name 2_ [2- (4, 4, -dimethoxytriphenylmethoxy) ethanesulfonyl) ethyl] Succinyl long-chain alkylamine-microporous glass beads (2- [2- (4, 4,-Dimethoxy tri tyloxy) ethylsulfomyl] ethyl-succinoyl long chain alkylamino-CPG)), synthesized on ABI 391EP DNA synthesizer The 3'-monophosphorylated oligonucleotide was obtained by removing the protecting group through concentrated ammonia. In order to show the stability of the 3-monophosphorylated oligonucleotide of the present invention to enzyme degradation, snake venom phosphodiesterase is used to represent the 3'-5 'exonuclease in the body, and DNase I is used to represent the endonuclease in the body. In vitro comparison of unmodified oligonucleotides, 3'-monophosphorylated oligonucleotides, thiophosphate-modified oligonucleotides, and y-partial thiophosphate-modified oligonucleotide pairs 3 '→ 5' exonucleic acid The stability of enzymes and endonucleases was further compared in serum and in cells. Experiments have shown that 3'-phosphorylated oligonucleotide fragments are resistant to snake venom phosphodiesterase, and the stability in serum and cells is significantly higher than that of 3 'unmodified or y-partial phosphorothioate-modified Nucleotide, slightly higher than phosphorothioate-modified oligonucleotide. Moreover, the 3'-phosphorylation modification does not affect the rate at which the oligonucleotide enters the cell, which is superior to the phosphorothioate-modified oligonucleotide. The above results indicate that 3 '→ 5' exonuclease is mainly present in serum and cells, and the enzyme needs to provide a substrate with 3 '-0H when it acts. When the oligonucleotide 3' 0H group is phosphorylated After that, it cannot be used as a substrate for 3 '→ 5' exonuclease, so it can resist the effect of 3 → 5 'exonuclease, prolong the residence time in human serum, and can be more efficiently transported to target cells. in. Since 3'-monophosphorylated oligonucleotides have high stability in cells, they can more effectively bind to the complementary sequence of the gene of interest.

3' -单磷酸化寡核苷酸只有被磷酸单酯酶切去 3'磷酸后才能被 3'—5'外切核酸酶降解。 3' -单磷酸化寡核苷酸在血清中和细胞内稳定性高，表明在血清中和细胞内磷酸单酯酶活性不高。 3'-monophosphorylated oligonucleotides can be degraded by 3'-5 'exonuclease only after the 3' phosphate is cut off by a phosphodiesterase. The 3'-monophosphorylated oligonucleotide has high stability in serum and intracellular cells, indicating that the activity of phosphodiesterase in serum and intracellular cells is not high.

此外，由于 DNA复制时需要 3'为 0H的引物， 3' -单磷酸化寡核苷酸引物一旦与 DNA结合还能阻遏病毒 D 的复制或 RNA反转录。目前在使用的反义寡核苷酸中大多是硫磷酸修饰的、 3'为 0H的寡核苷酸，它没有阻遏病毒 DM复制或 RNA反转录的作用。 3' -单磷酸化寡核苷酸对正常的碱基配对没有影响，因此能够保证这种经修饰的寡核苷酸正确地、专一地与靶 DNA或 RM配对。概括而言，依据病毒或肿瘤基因等有害基因 DNA或 RNA特定顺序而设计的、 3' -单磷酸化寡核苷酸片段能够专一地与靶 DNA或 RM结合，阻遏病毒 DNA的复制、转录和翻译，也可阻遏人体内有害基因的转录和翻译，也可利用体内 RNaseH使被 3' -单磷酸化寡核苷酸结合的靶 RNA降解。因此，由于上述作用机制和优越的性能， 3' -单磷酸化寡核苷酸有更多的生物功能。另外针对乙型肝炎病毒设计的一种反义寡核苷酸的研究证明， 3' -单磷酸化修饰比不修饰的寡核苷酸对于乙型肝炎病毒基因表达的抑制作用强一倍以上。 In addition, since 3′-OH primers are required for DNA replication, 3′-monophosphorylated oligonucleotide primers, once bound to DNA, can also inhibit virus D replication or RNA reverse transcription. Most of the currently used antisense oligonucleotides are phosphorothioate-modified, 3 ', 0H, oligonucleotides, which do not inhibit viral DM replication or RNA reverse transcription. The 3'-monophosphorylated oligonucleotide has no effect on normal base pairing, and thus can ensure that the modified oligonucleotide is correctly and specifically paired with the target DNA or RM. In summary, a 3'-monophosphorylated oligonucleotide fragment designed based on a specific sequence of DNA or RNA of a harmful gene such as a virus or a tumor gene can specifically bind to target DNA or RM to block the replication and transcription of viral DNA And translation, can also suppress the transcription and translation of harmful genes in the human body, and can also use RNaseH in the body to be 3'-monophosphorylated Oligonucleotide-bound target RNA is degraded. Therefore, due to the above-mentioned mechanism of action and superior properties, 3'-monophosphorylated oligonucleotides have more biological functions. In addition, research on an antisense oligonucleotide designed for hepatitis B virus has shown that 3'-monophosphorylization has more than twice the inhibitory effect on hepatitis B virus gene expression than unmodified oligonucleotides.

本发明的优点：本发明中提供的 3' -单磷酸化寡核苷酸具有比硫磷酸修饰的寡核苷酸还要高的稳定性和容易被细胞摄取的优点。而且，由于磷酸基团为天然核酸中固有成份，因此釆用磷酸基团作为修饰基团并没有引入非天然的修饰成份，其代谢降解产物无任何毒副作用，因此，与其它化学修饰方法相比使用时更为安全，其综合性能优于目前应用最广的硫磷酸取代的寡核苷酸。 - 单磷酸化寡核苷酸能专一地与靶 DM或 RNA结合，阻遏病毒 DNA的复制、转录和翻译。因此， 3' -单磷酸化寡核苷酸有望成为一种临床上有效的药物。附图说明 Advantages of the present invention: The 3'-monophosphorylated oligonucleotide provided by the present invention has the advantages of being more stable and easier to be taken up by cells than the phosphorothioate-modified oligonucleotide. Moreover, since the phosphate group is an inherent component in natural nucleic acids, the use of the phosphate group as a modification group does not introduce non-natural modification components, and its metabolic degradation products do not have any toxic and side effects. Therefore, compared with other chemical modification methods It is safer to use and its overall performance is better than the most widely used thiophosphate-substituted oligonucleotides. -Monophosphorylated oligonucleotides can specifically bind to target DM or RNA, and inhibit replication, transcription and translation of viral DNA. Therefore, 3'-monophosphorylated oligonucleotides are expected to become a clinically effective drug. BRIEF DESCRIPTION OF THE DRAWINGS

图 1 四种寡核苷酸片段对蛇毒磷酸二酯酶降解的抗性 Figure 1 Resistance of four oligo fragments to snake venom phosphodiesterase degradation

图 2 四种寡核苷酸片段对 DNase I 降解的抗性 Figure 2 Resistance of four oligonucleotide fragments to degradation of DNase I

图 3 四种寡核苷酸片段在 40 %人血清中的稳定性 Figure 3 Stability of four oligonucleotide fragments in 40% human serum

图 4 四种寡核苷酸片段在 HeLa细胞内的稳定性 Figure 4 Stability of four oligonucleotide fragments in HeLa cells

图 5 四种寡核苷酸片段在 HeLa细胞浆中的浓度 Figure 5 Concentrations of four oligonucleotide fragments in HeLa cytoplasm

图 6 四种寡核苷酸片段在 HeLa细胞核内的浓度最佳实施例 Figure 6 Concentrations of four oligonucleotide fragments in the nucleus of HeLa cells

实施例 1 3 -单磷酸化寡核苷酸片段和其它实验用寡核苷酸片段的合成利用 ABI公司的 391EP DNA合成仪合成下列四种寡核苷酸片段，其 DM顺序结构及 3'端修饰情况如下： Example 1 Synthesis of 3-Monophosphorylated Oligonucleotide Fragments and Other Experimental Oligonucleotide Fragments The following four oligonucleotide fragments were synthesized using ABI's 391EP DNA Synthesizer, their DM sequence structure and 3 'end The modification is as follows:

① 5' d (ATAGGGGCAT) V ② 3'P: 5' d (ATAGGGGCAT) p Ύ ① 5 'd (ATAGGGGCAT) V ② 3'P: 5 'd (ATAGGGGCAT) p Ύ

③ SP: d (A.T.A.G.G.G.G.C.A.G.A) V ③ SP: d (A.T.A.G.G.G.G.C.A.G.A) V

④ 3SP: 5' d (TTGAGGATGGAGCCCTGGA.C.CA) ④ 3SP: 5 'd (TTGAGGATGGAGCCCTGGA.C.CA)

* 代表硫磷酸二酯键位置 * Represents the position of thiophosphate diester bond

第①条寡核苷酸的 y为 OH基团； Article ① y is an OH group;

第②条寡核苷酸的 3'为单磷酸基团； 3 ′ of the oligonucleotide ② is a monophosphate group;

第③条寡核苷酸为全部核苷酸之间通过硫磷酸二酯键相连，为 0H基团；第④条寡核苷酸的 3'端有三个硫磷酸二酯键，其余为磷酸二酯键， 3'为 0H基团. 第②条 3'-单磷酸化寡核苷酸使用 0.2 μ ιτιοΐε 3'磷酸固相柱（ 3'- phosphate CPG Glen Research 公司产品，全称 2 ― [ 2- ( 4,4， —二甲基三苯甲氧基）乙磺酰] 乙基-丁二酰长链烷基胺-微孔玻璃珠（ 2-[ 2- ( 4,4'- Dimethoxy trityloxy ) ethylsulfomyl ] ethyl-succinoyl long chain alkylamino-CPG)) , 在 ABI 391EP DM合成仪上用 0.2 mole合成顺序合成。第①条 3'为 0H的寡核苷酸用 0.2 μιηοΐε dT固相柱（Glen Research 产品），用相同的合成顺序合成。第③条、第④条用 dA固相柱（Glen Research 产品），用相同的合成顺序合成，只有在硫磷酸修饰的位置用硫化试剂替代氧化试剂（参见 Iyer, RT， et al. J. Org. Chem. 1990, 55, 4693-4699) , 其余均相同。合成结束后，取出桂有寡核苷酸的载体，用浓氨水 55°C 处理 15小时脱去保护基，吸出溶液，真空浓缩至干，重新溶解于 200 μ ΐ 50%甲酰胺中，经 7 mol/L 尿素- 20%聚丙烯酰胺凝胶电泳纯化，紫外灯下割带，重蒸水浸泡，透析去盐，浓缩后 -2(TC保存。测 A₂₆。，计算产物量。实施例 2 对蛇毒磷酸二酯酶降解的稳定性 The third oligonucleotide is connected through a phosphorothioate bond between all the nucleotides, and is a 0H group. The third oligonucleotide of the fourth oligonucleotide has three phosphorophosphate diester bonds, and the rest are phosphorodiphosphates. Ester bond, 3 'is a 0H group. Article ② 3'-monophosphorylated oligonucleotide uses 0.2 μ ιτιοΐε 3' phosphate solid phase column (3'-phosphate CPG Glen Research company product, full name 2 ― [2- (4,4, —Dimethyltriphenylmethoxy) ethanesulfonyl] ethyl-succinyl long-chain alkylamine-microporous glass beads (2- [2- (4,4'- Dimethoxy trityloxy) ethylsulfomyl] ethyl-succinoyl long chain alkylamino-CPG)) was synthesized on a ABI 391EP DM synthesizer using a 0.2 mole synthesis sequence. Oligonucleotides whose 3 ′ is 0H in Article ① were synthesized using a 0.2 μιηοΐε dT solid-phase column (Glen Research product) using the same synthesis sequence. Articles ③ and ④ were synthesized using a dA solid-phase column (Glen Research product), which was synthesized in the same synthetic sequence, and the sulfuric acid reagent was used instead of the oxidizing reagent at the position modified by thiophosphoric acid (see Iyer, RT, et al. J. Org Chem. 1990, 55, 4693-4699), the rest are the same. After the synthesis was completed, the carrier containing the oligonucleotide was removed, treated with concentrated ammonia water at 55 ° C for 15 hours to remove the protective group, the solution was aspirated, concentrated in vacuo to dryness, and re-dissolved in 200 μΐ 50% formamide. mol / L urea-20% polyacrylamide gel electrophoresis purification, band cutting under UV lamp, dipping in distilled water, dialysis to desalinize, and concentrate -2 (stored in TC. Measured A _26. , calculate the amount of product. Example 2 Stability to snake venom phosphodiesterase degradation

实施例 1中所得到的四种寡核苷酸片段（3'0H， 3'P, SP, 3SP)分别用 T4 多聚核苷酸激酶（T4 ΡΝΚ)在其 5'端标记上 ³²Ρ，取 50 pmol寡核苷酸片段，加 50 μ α [ γ -³²p] -ATP, 2单位 T4多聚核苷酸激酶， 1 μ 1 10 倍缓冲液，加双蒸 '水至 10 μ 1， 37 °C保温 1小时，同实施例 1一样，用聚丙烯酰胺凝胶电泳的方法纯化标记的寡核苷酸，分别加入未标记的寡核苷酸至终浓度为 5 μ ηι₀1/ί, 加蛇毒磷酸二酯酶至 100 μ U/ml, 缓冲液为 10 醒 ol/L Tr is- HC1， 50 誦 ol/L MgCl₂, 0. 1% 牛血清白蛋白（BSA)， pH 8. 0, 37 °C保温, 在 0、 0. 5、 1、 1. 5、 2、 4、 8、 12、 24小时各取 5 μ 1, 加等体积上样缓冲液（98% 甲酰胺， 10 ■ol/L EDTA, 0. 025% 二甲苯蓝 FF， 0. 025% 溴酚蓝）混匀，用 7 mol/L尿素- 20%聚丙烯酰胺凝胶电泳鉴定。结果显示， 3' -单磷酸化寡核苷酸、全硫磷酸修饰的寡核苷酸以及 3'端三个硫磷酸二酯键的寡核苷酸对于蛇毒磷酸二酯酶的抗性作用相近，它们对蛇毒磷酸二酯酶的抗性都比 3' -0H的寡核苷酸要高许多，表现出显著差异。见图 1。实施例 3 对 DNase I (内切核酸酶）降解的稳定性 The four oligonucleotide fragments (3'0H, 3'P, SP, 3SP) obtained in Example 1 were each using T4 Polynucleotide kinase (T4 ΡΝΚ) at its 5 'end with the ^{32 Ρ,} taking 50 pmol oligonucleotide fragment, add ^{50 μ α [γ - 32 p} ] -ATP, 2 units of T4 polynucleotide Kinase, 1 μ 1 10-fold buffer, add double distilled water to 10 μ 1, and incubate at 37 ° C for 1 hour. As in Example 1, the labeled oligonucleotide was purified by polyacrylamide gel electrophoresis. Add unlabeled oligonucleotides to a final concentration of 5 μ ηι ₀ 1 / ί, add snake venom phosphodiesterase to 100 μ U / ml, and the buffer solution is 10 ol / L Tr is- HC1, 50 ol / L MgCl ₂ , 0.1% bovine serum albumin (BSA), pH 8. 0, 37 ° C incubation, 0, 0.5, 1, 1.5, 2, 4, 8, 12, 24 hours Take 5 μl each, add an equal volume of loading buffer (98% formamide, 10 ol / L EDTA, 0.025% xylene blue FF, 0.025% bromophenol blue) and mix, using 7 mol / L urea-20% polyacrylamide gel electrophoresis identification. The results showed that 3'-monophosphorylated oligonucleotides, perthiophosphate-modified oligonucleotides, and three thiophosphate diester-bonded oligonucleotides at the 3 'end had similar resistance to snake venom phosphodiesterase. Their resistance to snake venom phosphodiesterase is much higher than that of 3 '-0H oligonucleotides, showing a significant difference. see picture 1. Example 3 Stability to DNase I (endonuclease) degradation

同实施例 2—样制得 ³²P标记的四种寡核苷酸，在其中分别加入未标记的寡核苷酸至终浓度为 5 μ mol/L, 加 DNase I至 100 U/ml, 缓冲液为 10 腿 ol/L Tris-HCl, 5 mraol/L MgCl₂, 0. 1% BSA, pH 8. 0 , 37 °C保温，在 0、 0. 5、 1、 1. 5、 2、 4、 8、 12、 24小时各取 5 μ 1, 加等体积上样缓冲液混匀，用 7 mol/L 尿素- 20%聚丙烯酰胺凝胶电泳鉴定。结果显示， 3' -单磷酸化寡核苷酸对于 DNase I降解的稳定性略低于全硫磷酸修饰的寡核苷酸，但明显高于 3' -OH寡核苷酸和 3'端三个硫磷酸二酯键的寡核苷酸，而其中 3'端三个硫磷酸二酯键的寡核苷酸对 DNase I的作用特别敏感。见图 2。实施例 4 在 40%人血清体系中的稳定性 Example 2 - the same sample labeled ^{with 32} P to prepare four oligonucleotides, which were added in the non-labeled oligonucleotide to a final concentration of 5 μ mol / L, DNase I was added to 100 U / ml, buffered The solution is 10-leg ol / L Tris-HCl, 5 mraol / L MgCl ₂ , 0.1% BSA, pH 8. 0, 37 ° C, and maintained at 0, 0.5, 1, 1.5, 2, 4 Take 5 μl each at 1, 8, 12, and 24 hours. Add an equal volume of loading buffer and mix. Use 7 mol / L urea-20% polyacrylamide gel electrophoresis for identification. The results showed that the stability of 3'-monophosphorylated oligonucleotides to DNase I degradation was slightly lower than that of perthiophosphate-modified oligonucleotides, but significantly higher than that of 3'-OH oligonucleotides and 3'-terminal three One thiophosphodiester-bonded oligonucleotide, and the three thiophosphate diester-linked oligonucleotides at the 3 'end are particularly sensitive to the effect of DNase I. See Figure 2. Example 4 Stability in a 40% human serum system

同实施例 2—样制得 ³²P标记的四种寡核苷酸，在其中分别加入未标记的寡核苷酸至终浓度为 5 μιηοΙ/L, 加人血清至终浓度为 40%, 37°C保温，在 0、 0.5、 1、 1.5、 2、 4、 8、 12、 24小时各取 5μ 1，加等体积上样缓冲液混匀，用 7 mol/L尿素- 20%聚丙烯酰胺凝胶电泳鉴定。结果显示， 3'-单磷酸化寡核苷酸稳定性最好，反应 24小时后还留有近一半的量，明显高于其他几种结构的寡核苷酸，， 3' -0H的寡核苷酸在此体系中稳定性最差。见图 3。实施例 5 在细胞内的稳定性 In the same manner as in Example 2-four kinds of ³² P-labeled oligonucleotides were prepared, and unlabeled oligos were respectively added thereto. Nucleotide to a final concentration of 5 μιηοΙ / L, add human serum to a final concentration of 40%, incubate at 37 ° C, and take 5 μl each at 0, 0.5, 1, 1.5, 2, 4, 8, 12, 24 hours Add an equal volume of loading buffer and mix with 7 mol / L urea-20% polyacrylamide gel electrophoresis for identification. The results showed that the 3'-monophosphorylated oligonucleotide had the best stability, and nearly half of the amount remained after 24 hours of reaction, which was significantly higher than that of other structural oligonucleotides. Nucleotides are the least stable in this system. See Figure 3. Example 5 Stability in cells

在 35mm 培养皿中接种 2 χ 10⁵ HeLa细胞，加 1.5ml细胞培养液（ DMEM 含 10%小牛血清），于 37°C5%C0₂生长到培养孔面积的 40% - 60%，吸去培养液，分别加入 200 μ 1 5, Ρ- ³²标记的 3'0H、 3'P、 SP 或 3SP转染混合液（含 4 μ 1 Lipofectin, 3'0H、 3'P、 SP或 3SP 0.2 μ mol/L)， 5 小时后，吸干转染混合液，加入 2 ml 含 10%小牛血清的 DMEM培养液， 37°C5 %C0₂继续培养，并于 5， 12， 24, 36, 48， 72小时消化细胞，离心沉淀细胞，用酸性溶液洗去细胞表面的寡核苷酸（参见 Gao WY, et al. J. Biol. Chera. 1990, 265, 20172-20178; Lappalainen K. et al. Biochim. Biophys. Acta, 1994， 1196， 201-208), 加 1 ml TES液 ( 20 mmol/L Tris-HCl pH 8.0， 10 mmol/L EDTA, 1% SDS) , 于室温 10 分钟裂解细胞，用有机混合溶剂（苯酚：氯仿：异戊醇 =50: 48: 2)抽提两次，力口 2倍无水乙醇沉淀 DNA，力口 10 μ 1蒸馏水溶解， 7mol/L尿素- 15%聚丙烯酰胺凝胶电泳，放射自显影，然后，做黑度扫描，并以寡核苷酸转染 5小时的时候， HeLa细胞中全长寡核苷酸的含量为 100%, ^较在不同培养时间后细胞中所保留的全长寡核苷酸的百分比。结果显示：在 HeLa 细胞中， 3'P的稳定性要明显高于 3SP及 3'0H， SP稳定性也较高，见图 4。实施例 6 在细胞内的分布 Inoculate 2 x 10 ⁵ HeLa cells in a 35mm petri dish, add 1.5 ml of cell culture solution (DMEM contains 10% calf serum), and grow to 37%-60% of the area of the culture well at 37 ° C 5% C0 ₂ and aspirate. culture medium were added 200 μ 1 5, Ρ- ³² labeled 3'0H, 3'P, SP or 3SP transfection mixture (containing 4 μ 1 Lipofectin, 3'0H, 3'P , SP or 3SP 0.2 μ mol / L), after 5 hours, aspirate the transfection mixture, add 2 ml of DMEM culture medium containing 10% calf serum, and continue incubation at 37 ° C and 5% C0 ₂ at 5, 12, 24, 36, 48 The cells were digested for 72 hours, the cells were pelleted by centrifugation, and the oligonucleotides on the cell surface were washed with an acidic solution (see Gao WY, et al. J. Biol. Chera. 1990, 265, 20172-20178; Lappalainen K. et al. Biochim. Biophys. Acta, 1994, 1196, 201-208), add 1 ml TES solution (20 mmol / L Tris-HCl pH 8.0, 10 mmol / L EDTA, 1% SDS), lyse the cells at room temperature for 10 minutes, and use Organic mixed solvents (phenol: chloroform: isoamyl alcohol = 50: 48: 2) were extracted twice, and the DNA was precipitated by 2 times anhydrous ethanol, dissolved by 10 μl distilled water, 7mol / L urea-15% polypropylene Amide gel electrophoresis Autoradiography, then, after scanning for blackness and transfection with oligonucleotides for 5 hours, the content of full-length oligonucleotides in HeLa cells was 100%, compared with those retained in cells after different culture times The percentage of full-length oligonucleotides. The results show that in HeLa cells, the stability of 3'P is significantly higher than that of 3SP and 3'0H, and the stability of SP is also higher, as shown in Figure 4. Example 6 Distribution in cells

在 12孔细胞培养板中接种 5 X 10⁴ HeLa细胞，加 0.5ml细胞培养液（DMEM 含 10°/。小牛血清），于 37°C, 5 % C0₂ 生长到培养孔面积的 40% -60%，吸去培养液，分别加入 100 μ ΐ 5'_³²Ρ标记的 3'0H、 3,P、 SP或 3SP转染混合液（含 4 μ 1 Lipofectin, 3'0H、 3,P、 SP或 3SP 0.2 μιηοΙ/L), 每个样品重复三份， 5 小时后加入 400 μ 1含 10%小牛血清的 DMEM培养液， 37°C5 % C0₂继续培养，并于 0， 2， 4， 8， 18, 24, 30小时吸去培养液，用 0.5mlPBS洗一次，再用 ΙΟΟμ Ι 胰酶消化 6分钟，然后加 100μ 1含 10%小牛血清的 DMEM, 细胞计数三次，取平均值， 5000转 /分离心 1分钟沉淀细胞，同实施例 5用酸性溶液洗去细胞表面的寡核苷酸然后分离细胞浆及细胞核（参见 Weintraub H, et al. Cell 1983, 32,1191-1203) , 胞浆及胞核分别测定同位素放射性强度。计算 3'0H， 3'P, SP 和 3SP于不同时间在胞浆及胞核中的含量，三次的重复数据取平均值。 3'0H， 3'P, SP和 3SP四种寡核苷酸在细胞浆和细胞核内的浓度见图 5 和图 6。在实验的条件下， 3'0H，和 3'P进细胞比 SP和 3SP快， SP进细胞较慢， 3SP最慢。除 3SP 较长以外， 3'0H与 3'P长度相同， SP多一个核苷酸。从实验结果可见， 3' -单磷酸化修饰并不影响其进细胞速度。 Inoculate 5 X 10 ⁴ HeLa cells in a 12-well cell culture plate, add 0.5 ml of cell culture solution (DMEM Contains 10 ° /. Calf serum), at 37 ° C, 5% C0 ₂ to 40% -60% of the area of the culture wells, aspirate the culture medium, and add 100 μ ΐ 5'_ ³² Ρ labeled 3'0H, 3, P , SP or 3SP transfection mixture (containing 4 μ 1 Lipofectin, 3'0H, 3, P, SP or 3SP 0.2 μιηοΙ / L), repeat each sample in triplicate, and add 400 μ 1 after 5 hours containing 10% DMEM medium bovine serum, 37 ° C5% C0 ₂ cultured, and the culture medium at 0, 2, 4, 8, 18, 24, 30 hours aspirated, washed once with 0.5mlPBS, then trypsinized ΙΟΟμ Ι 6 minutes, then add 100 μl of DMEM containing 10% calf serum, count the cells three times, take the average value, and 5,000 rpm / separate the cells for 1 minute to precipitate the cells. Wash the oligonucleotides on the cell surface with an acidic solution as in Example 5. The cytoplasm and nucleus were then separated (see Weintraub H, et al. Cell 1983, 32, 1191-1203), and the cytoplasm and nucleus were measured for isotopic radioactive intensity, respectively. Calculate the contents of 3'0H, 3'P, SP and 3SP in the cytoplasm and nucleus at different times, and average the three repeated data. Concentrations of 3'0H, 3'P, SP and 3SP in the cytoplasm and nucleus are shown in Figure 5 and Figure 6. Under experimental conditions, 3'0H, and 3'P entered cells faster than SP and 3SP, SP entered cells more slowly, and 3SP was the slowest. Except that 3SP is longer, 3'0H is the same length as 3'P, and SP is one more nucleotide. It can be seen from the experimental results that the 3'-monophosphorylation modification does not affect its cell entry speed.

Claims

权利要求 Rights request

1.一种 3'-单磷酸化寡核苷酸，其特征在于其结构式可表示为： 1. A 3'-monophosphorylated oligonucleotide, characterized in that its structural formula can be expressed as:

5'd (丽 N……画） ρ3'或 oligo(dN)- 3'P, 5'd (Li N ... painting) ρ3 'or oligo (dN)-3'P,

式中 N = A、 G、 C、 T; Where N = A, G, C, T;

ρ3'或 3'P= 3'为单磷酸基团。 ρ3 'or 3'P = 3' is a monophosphate group.

2.按权利要求 1 所述的 3'-单磷酸化寡核苷酸的应用，其特征在于该寡核苷酸与靶 DNA或 RNA结合，阻遏病毒或有害基因的复制、转录和翻译，作为治疗的药物。 The use of the 3'-monophosphorylated oligonucleotide according to claim 1, characterized in that the oligonucleotide binds to target DNA or RNA, and inhibits the replication, transcription and translation of viruses or harmful genes, as Drugs for treatment.