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CN118460648A - A preparation method of QPI-1002 - Google Patents

A preparation method of QPI-1002 Download PDF

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CN118460648A
CN118460648A CN202410921728.1A CN202410921728A CN118460648A CN 118460648 A CN118460648 A CN 118460648A CN 202410921728 A CN202410921728 A CN 202410921728A CN 118460648 A CN118460648 A CN 118460648A
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substrate fragment
sense strand
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CN118460648B (en
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洪浩
詹姆斯·盖吉
张娜
焦学成
王磊
冯骏晨
张冉冉
胡守俊
蒋相军
陈仁芳
贾旭
李少贺
王吉忠
严思堂
金星
刘永贤
王思源
傅绪飞
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Tianjin Kailaiying Biotechnology Co ltd
Asymchem Laboratories Tianjin Co Ltd
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Abstract

The invention provides a preparation method of QPI-1002. The QPI-1002 is a double stranded RNA consisting of a sense strand and an antisense strand that are complementarily paired; the preparation method comprises the following steps: mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the sense strand substrate fragment is capable of constituting a sense strand and the antisense strand substrate fragment is capable of constituting an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, and the head base and the tail base of the sense strand substrate fragment and the antisense strand substrate fragment are not connected with each other to form a double-stranded nucleotide structure containing the nicks; the bases at both ends of the nick are linked by phosphodiester bonds using RNA ligase to form QPI-1002. Can solve the problem of lower purity of QPI-1002 prepared in the prior art, and is suitable for the technical field of pharmaceutical biosynthesis.

Description

一种QPI-1002的制备方法A preparation method of QPI-1002

技术领域Technical Field

本发明涉及药物生物合成领域，具体而言，涉及一种QPI-1002的制备方法。The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing QPI-1002.

背景技术Background Art

小干扰RNA(Small interfering RNA, siRNA)是长度为19-25nt的双链RNA，siRNA进入细胞后能够解离成单链，其中正义链通过碱基匹配能够同靶基因的信使RNA(messenger RNA, mRNA)特异性结合，诱发一系列作用，最终降解靶基因的mRNA，阻止mRNA的翻译以达到阻碍靶基因表达效果。近年来siRNA药物的研发获得的广泛的关注，siRNA药物的作用于mRNA，可成药的靶点要显著大于作用位点是蛋白质的传统小分子药物；此外，siRNA药物可以通过变换序列而作用于新的靶点，研发时间相对较短。Small interfering RNA (siRNA) is a double-stranded RNA with a length of 19-25 nt. After entering the cell, siRNA can dissociate into single strands, in which the positive strand can specifically bind to the messenger RNA (mRNA) of the target gene through base matching, inducing a series of effects, and finally degrading the mRNA of the target gene, preventing the translation of mRNA to achieve the effect of hindering the expression of the target gene. In recent years, the research and development of siRNA drugs has received widespread attention. siRNA drugs act on mRNA, and the druggable targets are significantly larger than traditional small molecule drugs whose action sites are proteins; in addition, siRNA drugs can act on new targets by changing the sequence, and the research and development time is relatively short.

QPI-1002是Quark公司开发的siRNA类双链RNA药物，目前处于临床3期实验阶段。QPI-1002可用于肾脏相关的疾病，例如抑制主要肾脏不良事件(Major Adverse KidneyEvents，MAKE)。QPI-1002 is a siRNA double-stranded RNA drug developed by Quark, and is currently in Phase 3 clinical trials. QPI-1002 can be used for kidney-related diseases, such as suppressing major adverse kidney events (MAKE).

目前，制备QPI-1002的方法为化学法，使用固相载体如可控微孔玻璃珠(Controlled Pore Glass, CPG)或聚苯乙烯树脂，通过亚磷酰胺三酯法循环合成，使寡核苷酸链沿3’至5’方向延伸，当合成循环结束后，通过氨解将QPI-1002链从固相载体切除，再经过纯化获得纯品。固相合成法为循环方法，合成的收率随着合成链长的增加而降低，而合成过程产生的杂质，如比目标序列多一个核苷酸的杂质(N+1杂质)或比目标序列少一个核苷酸的杂质(N-1杂质)，也会随着合成链长的增加而增多，此种杂质不易去除，导致纯化过程复杂，效率低下。因此QPI-1002制备的成本高昂，难以放大生产，因此需要开发一种更为高效的QPI-1002合成方法。At present, the method for preparing QPI-1002 is a chemical method, using a solid phase carrier such as controlled pore glass beads (CPG) or polystyrene resin, and cyclic synthesis through the phosphoramidite triester method to extend the oligonucleotide chain from 3' to 5'. After the synthesis cycle is completed, the QPI-1002 chain is cut off from the solid phase carrier by aminolysis, and then purified to obtain a pure product. The solid phase synthesis method is a cyclic method. The yield of the synthesis decreases with the increase of the length of the synthesis chain, and the impurities generated during the synthesis process, such as impurities with one more nucleotide than the target sequence (N+1 impurities) or impurities with one less nucleotide than the target sequence (N-1 impurities), will also increase with the increase of the length of the synthesis chain. Such impurities are not easy to remove, resulting in a complicated purification process and low efficiency. Therefore, the cost of preparing QPI-1002 is high and it is difficult to scale up production. Therefore, it is necessary to develop a more efficient QPI-1002 synthesis method.

发明内容Summary of the invention

本发明的主要目的在于提供一种QPI-1002的制备方法，以解决现有技术中制备获得的QPI-1002纯度较低的问题。The main purpose of the present invention is to provide a method for preparing QPI-1002 to solve the problem of low purity of QPI-1002 prepared in the prior art.

为了实现上述目的，根据本发明的一个方面，提供了一种QPI-1002的制备方法，该QPI-1002为由互补配对的正义链和反义链组成的双链RNA；该制备方法包括：将正义链底物片段、反义链底物片段和RNA连接酶混合，其中，正义链底物片段能够组成正义链，反义链底物片段能够组成反义链；正义链底物片段和反义链底物片段以碱基互补形成的氢键连接，正义链底物片段和反义链底物片段的头尾碱基之间均未互相连接，形成含有缺刻的双链核苷酸结构；利用RNA连接酶将缺刻两端的碱基以磷酸二酯键连接，形成QPI-1002；缺刻两端的碱基分别为不同底物片段的5’端和3’端，5’为磷酸根，3’端为羟基；利用RNA连接酶连接缺刻上下游的5’端的磷酸根和3’端的羟基，形成磷酸二酯键，获得QPI-1002；RNA连接酶包括RNA连接酶家族1和\或RNA连接酶家族2的RNA连接酶；优选地，RNA连接酶家族1的RNA连接酶为SEQ ID NO：5所示的RNA连接酶；RNA连接酶家族2的RNA连接酶选自：SEQ ID NO：1、SEQID NO：2、SEQ ID NO：3或SEQ ID NO：4所示的RNA连接酶中的一种或多种；或与SEQ ID NO：1~SEQ ID NO：5中所示的任一RNA连接酶具有70%以上同一性，且具有催化磷酸二酯键形成活性的酶。In order to achieve the above object, according to one aspect of the present invention, a method for preparing QPI-1002 is provided, wherein QPI-1002 is a double-stranded RNA composed of a complementary paired sense strand and an antisense strand; the preparation method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, so as to form a double-stranded RNA. A double-stranded nucleotide structure with a nick; using RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form QPI-1002; the bases at both ends of the nick are respectively the 5' end and the 3' end of different substrate fragments, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; using RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain QPI-1002; the RNA ligase includes RNA ligase family 1 and\or RNA ligase family 2 RNA ligase; preferably, the RNA ligase of RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; the RNA ligase of RNA ligase family 2 is selected from: one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or an enzyme that has more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5 and has activity in catalyzing the formation of phosphodiester bonds.

进一步地，正义链的核苷酸序列为具有SEQ ID NO：23所示的核苷酸序列的多聚核苷酸，反义链的核苷酸序列为具有SEQ ID NO：24所示的核苷酸序列的多聚核苷酸。Furthermore, the nucleotide sequence of the sense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 24.

进一步地，正义链底物片段包括2条或更多条，反义链底物片段包括2条或更多条；优选地，正义链底物片段的长度为5-12nt，更优选为6-11nt；优选地，反义链底物片段的长度为2-16nt，更优选为3-14nt。Furthermore, the sense strand substrate fragment includes 2 or more strands, and the antisense strand substrate fragment includes 2 or more strands; preferably, the length of the sense strand substrate fragment is 5-12nt, more preferably 6-11nt; preferably, the length of the antisense strand substrate fragment is 2-16nt, more preferably 3-14nt.

进一步地，正义链底物片段和反义链底物片段均包括2条，正义链底物片段包括第一正义链底物片段和第二正义链底物片段，反义链底物片段包括第一反义链底物片段和第二反义链底物片段；制备方法包括：S1）将第一正义链底物片段、第二正义链底物片段、第一反义链底物片段、第二反义链底物片段和RNA连接酶混合；S2）在RNA连接酶的催化作用下，第一正义链底物片段和第二正义链底物片段连接形成正义链，催化第一反义链底物片段和第二反义链底物片段连接形成反义链，正义链和反义链通过碱基互补配对形成QPI-1002；优选地，将正义链底物片段和反义链底物片段退火后与RNA连接酶混合，获得QPI-1002；优选地，第一正义链底物片段和第一反义链底物片段退火形成第一双链RNA片段，第二正义链底物片段和第二反义链底物片段退火形成第二双链RNA片段，第一双链RNA片段和第二双链RNA片段中均有≥3nt的碱基互补配对；优选地，第一双链RNA片段和第二双链RNA片段之间能够形成粘性末端，粘性末端的长度≥2nt。Further, the sense strand substrate fragment and the antisense strand substrate fragment each include 2 strands, the sense strand substrate fragment includes a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragment includes a first antisense strand substrate fragment and a second antisense strand substrate fragment; the preparation method includes: S1) mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment and RNA ligase; S2) under the catalytic action of RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form a sense strand, and the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to connect to form an antisense strand, and the sense strand substrate fragment and the antisense strand substrate fragment are connected to form an antisense strand. The sense strand and the antisense strand form QPI-1002 through base complementary pairing; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, the first sense strand substrate fragment and the first antisense strand substrate fragment are annealed to form a first double-stranded RNA fragment, and the second sense strand substrate fragment and the second antisense strand substrate fragment are annealed to form a second double-stranded RNA fragment, and the first double-stranded RNA fragment and the second double-stranded RNA fragment both have ≥3nt of base complementary pairing; preferably, the first double-stranded RNA fragment and the second double-stranded RNA fragment can form a sticky end, and the length of the sticky end is ≥2nt.

进一步地，S1中），第一正义链底物片段的核苷酸序列为SEQ ID NO：9所示的核苷酸序列，第二正义链底物片段的核苷酸序列为SEQ ID NO：10所示的核苷酸序列；优选地，第一反义链底物片段的核苷酸序列SEQ ID NO：12所示的核苷酸序列，第二反义链底物片段的核苷酸序列SEQ ID NO：11序列所示的核苷酸序列。Further, in S1), the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 11.

进一步地，S1中），第一正义链底物片段的核苷酸序列SEQ ID NO：13所示的核苷酸序列，第二正义链底物片段的核苷酸序列SEQ ID NO：14序列所示的核苷酸序列；优选地，第一反义链底物为SEQ ID NO：16所示的核苷酸序列，第二反义链底物为SEQ ID NO：15所示的核苷酸序列。Further, in S1), the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; preferably, the first antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 16, and the second antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 15.

进一步地，S1中），第一正义链底物片段的3’端与第二正义链底物片段的5’端在RNA连接酶的催化下连接，形成正义链；第一反义链底物片段的3’端与第二反义链底物片段的5’端在RNA连接酶的催化下连接，形成反义链；优选地，第一正义链底物片段的5’端为羟基基团，3’端为羟基基团；第二正义链底物片段的5’端为磷酸基团，3’端为羟基基团；优选地，第一反义链底物片段的5’端为羟基基团，3’端为羟基基团；第二反义链底物片段的5’端为磷酸基团，3’端为羟基基团。Further, in S1), the 3' end of the first sense chain substrate fragment and the 5' end of the second sense chain substrate fragment are connected under the catalysis of RNA ligase to form a sense chain; the 3' end of the first antisense chain substrate fragment and the 5' end of the second antisense chain substrate fragment are connected under the catalysis of RNA ligase to form an antisense chain; preferably, the 5' end of the first sense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group; preferably, the 5' end of the first antisense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

进一步地，正义链底物片段和反义链底物片段均包括3条，正义链底物片段包括第一正义链底物片段、第二正义链底物片段和第三正义链底物片段；反义链底物片段包括第一反义链底物片段、第二反义链底物片段和第三反义链底物片段；优选地，制备方法包括：S1）将第一正义链底物片段、第二正义链底物片段、第三正义链底物片段、第一反义链底物片段、第二反义链底物片段、第三反义链底物片段和RNA连接酶混合；S2）在RNA连接酶的催化作用下，第一正义链底物片段和第二正义链底物片段连接形成正义链，催化第一反义链底物片段和第二反义链底物片段连接形成反义链，正义链和反义链通过碱基互补配对形成QPI-1002；优选地，将正义链底物片段和反义链底物片段退火后与RNA连接酶混合，获得QPI-1002；优选地，S1）中，第一正义链底物片段的核苷酸序列为SEQ ID NO：17所示的核苷酸序列；第二正义链底物片段的核苷酸序列为SEQ ID NO：18所示的核苷酸序列；第三正义链底物片段的核苷酸序列为SEQ ID NO：19所示的核苷酸序列；优选地，S1）中，第一反义链底物片段的核苷酸序列为SEQ ID NO：20所示的核苷酸序列；第二反义链底物片段的核苷酸序列为SEQ ID NO：21所示的核苷酸序列；第三反义链底物片段的核苷酸序列为SEQ ID NO：22所示的核苷酸序列。Further, the sense strand substrate fragment and the antisense strand substrate fragment each include 3, the sense strand substrate fragment includes a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment; the antisense strand substrate fragment includes a first antisense strand substrate fragment, a second antisense strand substrate fragment and a third antisense strand substrate fragment; preferably, the preparation method comprises: S1) mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment and RNA ligase is mixed; S2) under the catalytic action of RNA ligase, the first sense chain substrate fragment and the second sense chain substrate fragment are connected to form a sense chain, and the first antisense chain substrate fragment and the second antisense chain substrate fragment are catalyzed to connect to form an antisense chain, and the sense chain and the antisense chain form QPI-1002 through base complementary pairing; preferably, the sense chain substrate fragment and the antisense chain substrate fragment are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, in S1), the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; preferably, in S1), the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 22.

进一步地，正义链底物片段及反义链底物片段浓度各自独立选自0.1-4.5mM，优选为0.8-1.6mM；优选地，正义链底物片段、反义链底物片段和RNA连接酶混合形成的反应体系中，还包括ATP、Tris-HCl、MgCl2和DTT；优选地，制备方法的反应温度为0℃~60℃，更优选为4℃~37℃；优选地，制备方法的反应时间为0.5h~24h，更优选为12-16h；优选地，制备方法的pH为6~8.5。Furthermore, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1-4.5 mM, preferably 0.8-1.6 mM; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase also includes ATP, Tris-HCl, MgCl 2 and DTT; preferably, the reaction temperature of the preparation method is 0°C~60°C, more preferably 4°C~37°C; preferably, the reaction time of the preparation method is 0.5h~24h, more preferably 12-16h; preferably, the pH of the preparation method is 6~8.5.

应用本发明的技术方案，利用上述制备方法，在RNA连接酶的催化下，正义链底物片段之间连接形成QPI-1002正义链，反义链底物片段之间连接形成QPI-1002反义链，从而实现了利用生物合成的方式制备此种siRNA药物。与化学合成制备QPI-1002的方法相比，本申请的制备方法获得的产物纯度高，产生的杂质少，制备过程简易，且反应条件温和，有机试剂用量低，降低生产成本，便于实现规模放大的工业化生产。By applying the technical solution of the present invention and utilizing the above-mentioned preparation method, under the catalysis of RNA ligase, the sense strand substrate fragments are connected to form the QPI-1002 sense strand, and the antisense strand substrate fragments are connected to form the QPI-1002 antisense strand, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the method of preparing QPI-1002 by chemical synthesis, the product obtained by the preparation method of the present application has high purity, few impurities, simple preparation process, mild reaction conditions, low amount of organic reagents, reduced production costs, and is easy to achieve scale-up industrial production.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

图1示出了根据本发明实施例1的酶催化连接反应示意图。FIG. 1 shows a schematic diagram of an enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

图2示出了根据本发明实施例1的RNA连接酶Ligase 31、Ligase 25和Ligase 11催化产物的电泳结果图。FIG. 2 shows the electrophoresis results of the catalytic products of RNA ligases Ligase 31, Ligase 25 and Ligase 11 according to Example 1 of the present invention.

图3示出了根据本发明实施例2的RNA连接酶Ligase 25催化产物的HPLC检测结果图。FIG. 3 shows a graph showing the HPLC detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention.

图4示出了根据本发明实施例2的RNA连接酶Ligase 25催化产物的LC-MS检测结果图。FIG. 4 shows the LC-MS detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention.

具体实施方式DETAILED DESCRIPTION

需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

术语解释：Terminology explanation:

N+1杂质：相较于目标合成序列具有单个核苷酸额外连接的核酸杂质。N+1 impurity: A nucleic acid impurity that has a single nucleotide extra attached compared to the target synthetic sequence.

N-1杂质：相较于目标合成序列具有单个核苷酸缺失的核酸杂质。N-1 impurity: A nucleic acid impurity that has a single nucleotide missing compared to the target synthetic sequence.

如背景技术所提到的，现有技术对于QPI-1002的制备采用化学合成的制备方法进行，不仅过程复杂，成本过高，而且产生的N+1和N-1杂质较多，影响后续对于产物的纯化。在本申请中发明人尝试开发一种QPI-1002的制备方法，利用酶催化合成对QPI-1002进行制备，因而提出了本申请的一系列保护方案。As mentioned in the background art, the prior art uses a chemical synthesis method for the preparation of QPI-1002, which is not only complicated and costly, but also produces a large number of N+1 and N-1 impurities, which affect the subsequent purification of the product. In this application, the inventors attempt to develop a method for preparing QPI-1002, using enzyme-catalyzed synthesis to prepare QPI-1002, and thus propose a series of protection schemes of this application.

在本申请第一种典型的实施方式中，提供了一种QPI-1002的制备方法，该QPI-1002为由互补配对的正义链和反义链组成的双链RNA；该制备方法包括：将正义链底物片段、反义链底物片段和RNA连接酶混合，其中，正义链底物片段能够组成正义链，反义链底物片段能够组成反义链；正义链底物片段和反义链底物片段以碱基互补形成的氢键连接，正义链底物片段和反义链底物片段的头尾碱基之间均未互相连接，形成含有缺刻的双链核苷酸结构；利用RNA连接酶将缺刻两端的碱基以磷酸二酯键连接，形成QPI-1002；缺刻两端的碱基分别为不同底物片段的5’端和3’端，5’为磷酸根，3’端为羟基；利用RNA连接酶连接缺刻上下游的5’端的磷酸根和3’端的羟基，形成磷酸二酯键，获得QPI-1002；RNA连接酶包括RNA连接酶家族1和\或RNA连接酶家族2的RNA连接酶；优选地，RNA连接酶家族1的RNA连接酶为SEQ ID NO：5所示的RNA连接酶；RNA连接酶家族2的RNA连接酶选自：SEQ ID NO：1、SEQ IDNO：2、SEQ ID NO：3或SEQ ID NO：4所示的RNA连接酶中的一种或多种；或与SEQ ID NO：1~SEQ ID NO：5中所示的任一RNA连接酶具有70%以上同一性，且具有催化磷酸二酯键形成活性的酶。In a first typical embodiment of the present application, a method for preparing QPI-1002 is provided, wherein QPI-1002 is a double-stranded RNA composed of a complementary sense strand and an antisense strand; the preparation method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, so as to form a double-stranded RNA containing a missing strand. The invention relates to a double-stranded nucleotide structure of the nick; using RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form QPI-1002; the bases at both ends of the nick are the 5' end and the 3' end of different substrate fragments, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; using RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain QPI-1002; the RNA ligase includes RNA ligase family 1 and/or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; the RNA ligase of RNA ligase family 2 is selected from: one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or an enzyme having more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5 and having activity of catalyzing the formation of phosphodiester bonds.

在上述的制备方法中，正义链底物片段为能够组成正义链的2条或更多条的核苷酸序列，即正义链底物片段的多条核苷酸序列可拼接组成与正义链序列相同的序列，与正义链的区别在于正义链底物片段之间存在缺刻，未以磷酸二酯键相连接。同理，反义链底物片段及反义链具有上述的特征。利用RNA连接酶将2条或多条正义链底物片段或反义链底物片段之间以磷酸二酯键相连接，获得QPI-1002的正义链及反义链。In the above preparation method, the sense strand substrate fragment is a nucleotide sequence that can form two or more sense strands, that is, multiple nucleotide sequences of the sense strand substrate fragment can be spliced to form a sequence identical to the sense strand sequence, and the difference from the sense strand is that there are nicks between the sense strand substrate fragments and they are not connected by phosphodiester bonds. Similarly, the antisense strand substrate fragment and the antisense strand have the above characteristics. Two or more sense strand substrate fragments or antisense strand substrate fragments are connected by phosphodiester bonds using RNA ligase to obtain the sense strand and antisense strand of QPI-1002.

在上述的制备方法中，将正义链底物片段、反义链底物片段与RNA连接酶混合，能够制备获得QPI-1002。在此制备方法中，正义链底物片段及反义链底物片段之间能够进行互补配对，形成含有粘性末端的双链结构的核苷酸，该含有粘性末端的双链结构核苷酸继续与其他底物片段结合，形成含有缺刻的双链核苷酸，RNA连接酶可识别此种双链结构的缺刻，以磷酸二酯键对缺刻进行连接，从而制备获得目标产物QPI-1002。In the above preparation method, the sense strand substrate fragment, the antisense strand substrate fragment and RNA ligase are mixed to prepare QPI-1002. In this preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be complementary paired to form a double-stranded nucleotide containing a sticky end, and the double-stranded nucleotide containing a sticky end continues to bind to other substrate fragments to form a double-stranded nucleotide containing a nick, and RNA ligase can recognize the nick of this double-stranded structure and connect the nick with a phosphodiester bond, thereby preparing the target product QPI-1002.

在一种优选的实施例中，正义链的核苷酸序列为具有SEQ ID NO：23所示的核苷酸序列的多聚核苷酸，反义链的核苷酸序列为具有SEQ ID NO：24所示的核苷酸序列的多聚核苷酸。In a preferred embodiment, the nucleotide sequence of the sense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 24.

SEQ ID NO：23：SEQ ID NO: 23:

GAmGAmAUmAUmUUmCAmCCmCUmUCmA。GAmGAmAUmAUmUUmCAmCCmCUmUCmA.

SEQ ID NO：24：SEQ ID NO: 24:

UmGAmAGmGGmUGmAAmAUmAUmUCmUCm。UmGAmAGmGGmUGmAAmAUmAUmUCmUCm.

本申请中，A、C、G或U后的m表述对该核糖核苷酸的2’甲氧基修饰。In the present application, m after A, C, G or U represents the 2' methoxy modification of the ribonucleotide.

在一种优选的实施例中，RNA连接酶包括RNA连接酶家族1和\或RNA连接酶家族2的RNA连接酶；优选地，RNA连接酶家族1的RNA连接酶为SEQ ID NO：5所示的RNA连接酶；RNA连接酶家族2的RNA连接酶选自：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示的RNA连接酶中的一种或多种；或与SEQ ID NO：1~SEQ ID NO：5中所示的任一RNA连接酶具有70%以上同一性，包括但不限于75%、80%、85%、90%、95%、99%以上（比如85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、98.5%、99%、99.5%、99.6%、99.7%、99.8%以上，甚至99.9%以上）且具有催化磷酸二酯键形成活性的酶。In a preferred embodiment, the RNA ligase includes RNA ligases of RNA ligase family 1 and/or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; the RNA ligase of RNA ligase family 2 is selected from: one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or an enzyme having more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) and having activity in catalyzing phosphodiester bond formation.

上述RNA连接酶均能够识别底物片段互补配对形成的、带有缺刻的双链结构，从而实现催化磷酸基团与羟基基团形成磷酸二酯键。The above-mentioned RNA ligases are all able to recognize the double-stranded structure with nicks formed by complementary pairing of substrate fragments, thereby catalyzing the formation of phosphodiester bonds between phosphate groups and hydroxyl groups.

SEQ ID NO：1（Ligase 25，Vibrio phage NT-1）：SEQ ID NO: 1 (Ligase 25, Vibrio phage NT-1):

MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF。MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSE KRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.

SEQ ID NO：2（Ligase 26，Escherichia phage AR1）：SEQ ID NO: 2 (Ligase 26, Escherichia phage AR1):

MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY。MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVE EAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY.

SEQ ID NO：3（Ligase 31，Vibrio phage VH12019）：SEQ ID NO: 3 (Ligase 31, Vibrio phage VH12019):

MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAHADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA。MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAH ADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA.

SEQ ID NO：4:（Ligase 41，Vibrio phage VH7D）：SEQ ID NO: 4: (Ligase 41, Vibrio phage VH7D):

MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATAHEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV。MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATA HEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV.

SEQ ID NO：5（Ligase 42，Escherichia phage JN02）：SEQ ID NO: 5 (Ligase 42, Escherichia phage JN02):

MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY。MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDV KSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY.

SEQ ID NO：6：（Ligase 11，Thermococcus）：SEQ ID NO: 6: (Ligase 11, Thermococcus):

MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD。MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKV GELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.

SEQ ID NO：7：（Ligase 20，Archaea）：SEQ ID NO: 7: (Ligase 20, Archaea):

MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH。MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGS GNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQV ASDYLRIMERAIRKYRIPWPDRELLVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYK NVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.

SEQ ID NO：8：（Ligase 32，Bacteria）：SEQ ID NO: 8: (Ligase 32, Bacteria):

MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD。MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDE LYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.

本申请中的同一性（Identity）是指氨基酸序列或核苷酸序列之间的“同一性”，即氨基酸序列或核苷酸序列中的种类相同的氨基酸残基或核苷酸的比率的总计。氨基酸序列或核苷酸序列的同一性可以利用BLAST（Basic Local Alignment Search Tool）、FASTA等比对程序来确定。Identity in this application refers to the "identity" between amino acid sequences or nucleotide sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

70%、75%、80%、85%、90%、95%、99%以上（比如85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、98.5%、99%、99.5%、99.6%、99.7%、99.8%以上，甚至99.9%以上）同一性且具有相同功能的蛋白质，其活性位点、活性口袋、活性机制、蛋白结构等均和上述序列提供的蛋白质大概率相同。Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are likely to be the same as the proteins provided by the above sequences.

如本文所用，氨基酸残基缩写如下：丙氨酸（Ala；A）、天冬酰胺（Asn；N）、天冬氨酸（Asp；D）、精氨酸（Arg；R）、半胱氨酸（Cys；C）、谷氨酸（Glu；E）、谷氨酰胺（Gln；Q）、甘氨酸（Gly；G）、组氨酸（His；H）、异亮氨酸（Ile；I）、亮氨酸（Leu；L）、赖氨酸（Lys；K）、蛋氨酸（Met；M）、苯丙氨酸（Phe；F）、脯氨酸（Pro；P），丝氨酸（Ser；S）、苏氨酸（Thr；T）、色氨酸（Trp；W）、酪氨酸（Tyr；Y）和缬氨酸（Val；V）。As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

取代、替换等规则，一般情况下，哪些氨基酸性质类似，替换后的效果也类似。例如，在上述同源蛋白中，可发生保守的氨基酸替换。“保守的氨基酸替换”包括但不限于：Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

疏水性氨基酸（Ala、Cys、Gly、Pro、Met、Val、Ile、Leu）被其他疏水性氨基酸取代；hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

侧链粗大的疏水性氨基酸（Phe、Tyr、Trp）被其他侧链粗大的疏水性氨基酸取代；Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

侧链带正电的氨基酸（Arg、His、Lys）被其他侧链带正电的氨基酸取代；Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

侧链有极性不带电的氨基酸（Ser、Thr、Asn、Gln）被其他侧链有极性不带电的氨基酸取代。Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

本领域技术人员也可以根据现有技术中的“blosum62评分矩阵”等本领域技术人员熟知的氨基酸替换规则对氨基酸进行保守替换。A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

本申请中只能通过SEQ ID NO：1~SEQ ID NO：5所示的RNA连接酶，或与SEQ ID NO：1~SEQ ID NO：5中所示的任一RNA连接酶具有70%以上同一性的酶催化本申请中底物的磷酸集团与羟基集团形成磷酸二酯键，获得产物QPI-1002。在本申请的相关实验中，发明人通过从大量的酶中进行筛选，获得了能够合成QPI-1002的上述SEQ ID NO：1~SEQ ID NO：5所示的RNA连接酶。而实验中占比极大的阴性结果显示大多数RNA连接酶难以催化QPI-1002的合成，包括但不限于SEQ ID NO：6~SEQ ID NO：8中所示的RNA连接酶，在本申请说明书只以SEQID NO：6~SEQ ID NO：8为例体现该类没有催化QPI-1002合成活性的RNA连接酶。In the present application, only the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5, or an enzyme with more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5, can be used to catalyze the formation of a phosphodiester bond between the phosphate group and the hydroxyl group of the substrate in the present application to obtain the product QPI-1002. In the relevant experiments of the present application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5 that can synthesize QPI-1002 by screening from a large number of enzymes. However, the negative results that accounted for a large proportion in the experiment showed that most RNA ligases were difficult to catalyze the synthesis of QPI-1002, including but not limited to the RNA ligases shown in SEQ ID NO: 6 to SEQ ID NO: 8. In the present application specification, only SEQ ID NO: 6 to SEQ ID NO: 8 are used as examples to reflect this type of RNA ligase that has no catalytic activity in the synthesis of QPI-1002.

在一种优选的实施例中，正义链底物片段包括2条或更多条，反义链底物片段包括2条或更多条；优选地，正义链底物片段的长度为5-12nt，更优选为6-11nt；优选地，反义链底物片段的长度为2-16nt，更优选为3-14nt。In a preferred embodiment, the sense strand substrate fragment includes 2 or more strands, and the antisense strand substrate fragment includes 2 or more strands; preferably, the length of the sense strand substrate fragment is 5-12nt, more preferably 6-11nt; preferably, the length of the antisense strand substrate fragment is 2-16nt, more preferably 3-14nt.

在一种优选的实施例中，正义链底物片段和反义链底物片段均包括2条，正义链底物片段包括第一正义链底物片段和第二正义链底物片段，反义链底物片段包括第一反义链底物片段和第二反义链底物片段；制备方法包括：S1）将第一正义链底物片段、第二正义链底物片段、第一反义链底物片段和第二反义链底物片段混合；S2）在RNA连接酶的催化作用下，第一正义链底物片段和第二正义链底物片段连接形成正义链，催化第一反义链底物片段和第二反义链底物片段连接形成反义链，正义链和反义链通过碱基互补配对形成QPI-1002；优选地，将正义链底物片段和反义链底物片段退火后与RNA连接酶混合，获得QPI-1002；优选地，第一正义链底物片段和第一反义链底物片段退火形成第一双链RNA片段，第二正义链底物片段和第二反义链底物片段退火形成第二双链RNA片段，第一双链RNA片段和第二双链RNA片段中均有≥3nt的碱基互补配对；优选地，第一双链RNA片段和第二双链RNA片段之间能够形成粘性末端，粘性末端的长度≥2nt。In a preferred embodiment, the sense strand substrate fragment and the antisense strand substrate fragment each include 2 strands, the sense strand substrate fragment includes a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragment includes a first antisense strand substrate fragment and a second antisense strand substrate fragment; the preparation method comprises: S1) mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment and the second antisense strand substrate fragment; S2) under the catalytic action of RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form a sense strand, and the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to connect to form an antisense strand, and the sense strand substrate fragment and the antisense strand substrate fragment are connected to form an antisense strand; The sense strand and the antisense strand form QPI-1002 through base complementary pairing; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, the first sense strand substrate fragment and the first antisense strand substrate fragment are annealed to form a first double-stranded RNA fragment, and the second sense strand substrate fragment and the second antisense strand substrate fragment are annealed to form a second double-stranded RNA fragment, and the first double-stranded RNA fragment and the second double-stranded RNA fragment both have ≥3nt of base complementary pairing; preferably, the first double-stranded RNA fragment and the second double-stranded RNA fragment can form a sticky end, and the length of the sticky end is ≥2nt.

在上述制备方法中，可以先将正义链底物片段和反义链底物片段进行混合进行退火，正义链底物片段和反义链底物片段之间能够通过碱基互补配对形成双链RNA结构，在该双链RNA结构中存在不同底物片段之间的缺刻。再将退火后的反应体系与RNA连接酶混合，利用RNA连接酶将缺刻两侧的磷酸基团和羟基基团以磷酸二酯键相连接，修复缺刻，从而获得双链结构完整的目标产物QPI-1002。In the above preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be mixed and annealed first, and the sense strand substrate fragment and the antisense strand substrate fragment can form a double-stranded RNA structure through base complementary pairing, and there are nicks between different substrate fragments in the double-stranded RNA structure. Then, the annealed reaction system is mixed with RNA ligase, and the RNA ligase is used to connect the phosphate groups and hydroxyl groups on both sides of the nick with phosphodiester bonds to repair the nick, thereby obtaining the target product QPI-1002 with a complete double-stranded structure.

在一种优选的实施例中，S1）中，第一正义链底物片段的核苷酸序列为SEQ ID NO：9所示的核苷酸序列，第二正义链底物片段的核苷酸序列为SEQ ID NO：10所示的核苷酸序列。优选地，第一反义链底物的核苷酸序列为SEQ ID NO：12所示的核苷酸序列，第二反义链底物的核苷酸序列为SEQ ID NO：11序列所示的核苷酸序列。In a preferred embodiment, in S1), the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10. Preferably, the nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 11.

在一种优选的实施例中，S1）中，第一正义链底物的核苷酸序列为SEQ ID NO：13所示的核苷酸序列，第二正义链底物的核苷酸序列为SEQ ID NO：14所示的核苷酸序列。优选地，第一反义链底物的核苷酸序列为SEQ ID NO：16所示的核苷酸序列，第二反义链底物的核苷酸序列为SEQ ID NO：15所示的核苷酸序列。In a preferred embodiment, in S1), the nucleotide sequence of the first sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 14. Preferably, the nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 15.

在一种优选的实施例中，S2）中，第一正义链底物片段的3’端与第二正义链底物片段的5’端在RNA连接酶的催化下连接，形成正义链；第一反义链底物片段的3’端与第二反义链底物片段的5’端在RNA连接酶的催化下连接，形成反义链；优选地，第一正义链底物片段的5’端为羟基基团，3’端为羟基基团；第二正义链底物片段的5’端为磷酸基团，3’端为羟基基团；优选地，第一反义链底物片段的5’端为羟基基团，3’端为羟基基团；第二反义链底物片段的5’端为磷酸基团，3’端为羟基基团。In a preferred embodiment, in S2), the 3' end of the first sense chain substrate fragment and the 5' end of the second sense chain substrate fragment are connected under the catalysis of RNA ligase to form a sense chain; the 3' end of the first antisense chain substrate fragment and the 5' end of the second antisense chain substrate fragment are connected under the catalysis of RNA ligase to form an antisense chain; preferably, the 5' end of the first sense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group; preferably, the 5' end of the first antisense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

利用上述制备方法和SEQ ID NO：9-SEQ ID NO：12或SEQ ID NO：13-SEQ ID NO：16所示的底物片段，能够制备QPI-1002。但需要说明的是，对于底物片段的选择并非仅限于上述SEQ ID NO：9-12或SEQ ID NO：13-16所示的底物片段，能够组合形成正义链和反义链的底物片段均能够应用于上述制备方法中，上述制备方法适用于QPI-1002的制备但不局限于底物片段连接位置的不同，上述制备对于QPI-1002的正义链序列和反义链序列的连接均具有较好的连接效果。正义链底物片段或反义链底物片段的数量包括但不限于2条、3条、4条乃至更多条。QPI-1002 can be prepared using the above preparation method and the substrate fragments shown in SEQ ID NO: 9-SEQ ID NO: 12 or SEQ ID NO: 13-SEQ ID NO: 16. However, it should be noted that the selection of substrate fragments is not limited to the substrate fragments shown in SEQ ID NO: 9-12 or SEQ ID NO: 13-16. All substrate fragments that can be combined to form a sense chain and an antisense chain can be applied to the above preparation method. The above preparation method is suitable for the preparation of QPI-1002 but is not limited to the different connection positions of the substrate fragments. The above preparation has a good connection effect for the connection of the sense chain sequence and the antisense chain sequence of QPI-1002. The number of sense chain substrate fragments or antisense chain substrate fragments includes but is not limited to 2, 3, 4 or even more.

SEQ ID NO：9：GAmGAmAUmAUmUUmCAmC。SEQ ID NO: 9: GAmGAmAUmAUmUUmCAmC.

SEQ ID NO：10：CmCUmUCmA。SEQ ID NO: 10: CmCUmUCmA.

SEQ ID NO：11：AUmAUmUCmUCm。SEQ ID NO: 11: AUmAUmUCmUCm.

SEQ ID NO：12：UmGAmAGmGGmUGmAAm。SEQ ID NO: 12: UmGAmAGmGGmUGmAAm.

SEQ ID NO：13：GAmGAmAUmAUm。SEQ ID NO: 13: GAmGAmAUmAUm.

SEQ ID NO：14：UUmCAmCCmCUmUCmA。SEQ ID NO: 14: UUmCAmCCmCUmUCmA.

SEQ ID NO：15：UmUCmUCm。SEQ ID NO: 15: UmUCmUCm.

SEQ ID NO：16：UmGAmAGmGGmUGmAAmAUmA。SEQ ID NO: 16: UmGAmAGmGGmUGmAAmAUmA.

在一种优选的实施例中，正义链底物片段和反义链底物片段均包括3条，正义链底物片段包括第一正义链底物片段、第二正义链底物片段和第三正义链底物片段；反义链底物片段包括第一反义链底物片段、第二反义链底物片段和第三反义链底物片段；优选地，制备方法包括：S1）将第一正义链底物片段、第二正义链底物片段、第三正义链底物片段、第一反义链底物片段、第二反义链底物片段、第三反义链底物片段和RNA连接酶混合；S2）在RNA连接酶的催化作用下，第一正义链底物片段和第二正义链底物片段连接形成正义链，催化第一反义链底物片段和第二反义链底物片段连接形成反义链，正义链和反义链通过碱基互补配对形成QPI-1002；优选地，将正义链底物片段和反义链底物片段退火后与RNA连接酶混合，获得QPI-1002；优选地，S1）中，第一正义链底物片段的核苷酸序列为SEQ ID NO：17所示的核苷酸序列；第二正义链底物片段的核苷酸序列为SEQ ID NO：18所示的核苷酸序列；第三正义链底物片段的核苷酸序列为SEQ ID NO：19所示的核苷酸序列；优选地，S1）中，第一反义链底物片段的核苷酸序列为SEQ ID NO：22所示的核苷酸序列；第二反义链底物片段的核苷酸序列为SEQ ID NO：21所示的核苷酸序列；第三反义链底物片段的核苷酸序列为SEQID NO：20所示的核苷酸序列。In a preferred embodiment, the sense strand substrate fragment and the antisense strand substrate fragment each include 3, the sense strand substrate fragment includes a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment; the antisense strand substrate fragment includes a first antisense strand substrate fragment, a second antisense strand substrate fragment and a third antisense strand substrate fragment; preferably, the preparation method comprises: S1) mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment The fragments are mixed with RNA ligase; S2) under the catalytic action of RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form a sense strand, and the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to connect to form an antisense strand, and the sense strand and the antisense strand are formed by base complementary pairing. QPI-1002; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, in S1), the nucleotide sequence of the first sense strand substrate fragment is SEQ The nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; preferably, in S1), the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 22; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20.

SEQ ID NO：17：GAmGAmAUm。SEQ ID NO: 17: GAmGAmAUm.

SEQ ID NO：18：AUmUUmCAmC。SEQ ID NO: 18: AUmUUmCAmC.

SEQ ID NO：19：CmCUmUCmA。SEQ ID NO: 19: CmCUmUCmA.

SEQ ID NO：20：CmUCm。SEQ ID NO: 20: CmUCm.

SEQ ID NO：21：AAmAUmAUmU。SEQ ID NO: 21: AAmAUmAUmU.

SEQ ID NO：22：UmGAmAGmGGmUGm。SEQ ID NO: 22: UmGAmAGmGGmUGm.

在一种优选的实施例中，正义链底物片段及反义链底物片段浓度各自选自为0.1-4.5mM，优选为0.8-1.6mM；优选地，正义链底物片段、反义链底物片段和RNA连接酶混合形成的反应体系中，还包括ATP、Tris-HCl、MgCl2和DTT；优选地，制备方法的反应温度为0℃~60℃，更优选为4℃~37℃；优选地，制备方法的反应时间为0.5h~24h，更优选为12-16h；优选地，制备方法的pH为6~8.5。In a preferred embodiment, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are each selected from 0.1-4.5 mM, preferably 0.8-1.6 mM; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase further comprises ATP, Tris-HCl, MgCl 2 and DTT; preferably, the reaction temperature of the preparation method is 0°C~60°C, more preferably 4°C~37°C; preferably, the reaction time of the preparation method is 0.5h~24h, more preferably 12-16h; preferably, the pH of the preparation method is 6~8.5.

上述正义链底物片段和反义链底物片段的浓度各自选自包括但不限于0.1、0.5、0.8、0.9、1、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.5、3.0、3.5、4.0或4.5mM；上述制备方法的反应温度包括但不限于10、15、16、20、25、30、35或40℃；上述制备方法的反应时间包括但不限于2、5、10、15、16、20、24、25、30、35、40、45或48h；上述制备方法的pH包括但不限于6、6.5、7、7.5、8或8.5。The concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are each selected from, including but not limited to, 0.1, 0.5, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5 mM; the reaction temperature of the preparation method includes but is not limited to 10, 15, 16, 20, 25, 30, 35 or 40°C; the reaction time of the preparation method includes but is not limited to 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45 or 48h; the pH of the preparation method includes but is not limited to 6, 6.5, 7, 7.5, 8 or 8.5.

下面将结合具体的实施例来进一步详细解释本申请的有益效果。The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

实施例1Example 1

基于QPI-1002的序列设计如表1所示的4个单链RNA片段，长度单位为nt。Four single-stranded RNA fragments were designed based on the sequence of QPI-1002 as shown in Table 1, and the length unit is nt.

表1Table 1

。 .

其中，A、C、G或U后的m表示对该核糖核苷酸的2’甲氧基修饰。The m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide.

底物1的第2、4、6、8、10和12位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4, 6, 8, 10 and 12 of substrate 1 have 2' methoxy modifications.

底物2的第1、3和5位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1, 3 and 5 of substrate 2 have 2' methoxy modifications.

底物3的第2、4、6和8位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4, 6 and 8 of substrate 3 have 2' methoxy modifications.

底物4的第1、3、5、7、9和11位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1, 3, 5, 7, 9 and 11 of substrate 4 have 2' methoxy modifications.

使用固相合成方法制备上述4个单链RNA片段。The above four single-stranded RNA fragments were prepared using a solid phase synthesis method.

将4个单链RNA片段以等摩尔比例混匀后得到终浓度2.5mM（每种底物均为2.5mM）的底物混合物，退火，获得退火后的RNA片段混合液。将退火后的RNA片段混合液进行酶催化连接反应，反应体系设置为10μL，反应体系中包括反应缓冲液(50mM Tris-HCl，pH 7.5)、三磷酸腺苷(adenosine triphosphate，ATP)、MgCl2、二硫苏糖醇(dithiothreitol，DTT)，分别加RNA连接酶Ligase 25、Ligase 26、Ligase 31、Ligase 41、Ligase 42、Ligase 11、Ligase 20和Ligase 32。将反应体系于16℃反应16h。得到的反应体系经80℃ 5min失活连接酶，经12000rpm离心去除沉淀。酶催化连接反应示意图如图1所示。The four single-stranded RNA fragments were mixed in an equimolar ratio to obtain a substrate mixture with a final concentration of 2.5 mM (each substrate was 2.5 mM), and annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzyme-catalyzed ligation reaction. The reaction system was set to 10 μL. The reaction system included a reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl 2 , dithiothreitol (DTT), and RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42, Ligase 11, Ligase 20, and Ligase 32 were added respectively. The reaction system was reacted at 16°C for 16 hours. The obtained reaction system was inactivated by heating the ligase at 80°C for 5 minutes, and the precipitate was removed by centrifugation at 12000 rpm. The schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1.

将RNA连接酶Ligase 25、Ligase 26、Ligase 31、Ligase 41、Ligase 42 Ligase11、Ligase 20和Ligase 32催化得到的产物进行SDS-PAGE检测。RNA连接酶Ligase31、Ligase25和Ligase11催化得到的产物电泳结果图如图2所示，图2中M泳道代表RNA分子标准（marker），1泳道表示Ligase31的反应体系，2泳道表示Ligase25的反应体系，3泳道表示Ligase 11的反应体系。产率根据Urea-PAGE结果中目标条带的灰度分析结果估算，最终产率结果如表2所示。The products catalyzed by RNA ligase Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42 Ligase11, Ligase 20 and Ligase 32 were detected by SDS-PAGE. The electrophoresis results of the products catalyzed by RNA ligase Ligase31, Ligase25 and Ligase11 are shown in Figure 2. In Figure 2, lane M represents the RNA molecule standard (marker), lane 1 represents the reaction system of Ligase31, lane 2 represents the reaction system of Ligase25, and lane 3 represents the reaction system of Ligase 11. The yield was estimated based on the grayscale analysis results of the target band in the Urea-PAGE results, and the final yield results are shown in Table 2.

对10种RNA连接酶进行活力筛选，发现Ligase25连接效果较好，能够将大部分底物转化为QPI-1002。Ten RNA ligases were screened for activity, and it was found that Ligase25 had a better ligation effect and could convert most substrates into QPI-1002.

制备获得的QPI-1002的正义链为GAmGAmAUmAUmUUmCAmCCmCUmUCmA（SEQ ID NO：23）；反义链为UmGAmAGmGGmUGmAAmAUmAUmUCmUCm（SEQ ID NO：24）。The sense strand of the prepared QPI-1002 is GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23); the antisense strand is UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).

反应结果如表2所示。The reaction results are shown in Table 2.

表2Table 2

。 .

备注：Remark:

1）反应条件：底物片段100μM，ATP 10eq，MgCl2 100eq，DTT 10eq（1eq=100μM），终浓度为0.2mg/mL酶，2385V、50 mM Tris-HCl pH 7.5，16℃，16 h；1) Reaction conditions: substrate fragment 100 μM, ATP 10 eq, MgCl 2 100 eq, DTT 10 eq (1 eq = 100 μM), final concentration 0.2 mg/mL enzyme, 2385 V, 50 mM Tris-HCl pH 7.5, 16 °C, 16 h;

2）N.D.表示未检测到产品生成，++表示25~50%（不包括50%的端点值），+++表示50~75%，++++表示>75%。2) N.D. means no product formation was detected, ++ means 25~50% (excluding the endpoint value of 50%), +++ means 50~75%, and ++++ means >75%.

通过Urea-PAGE凝胶电泳结果图片的灰度分析得到的产品和底物的灰度数据，此实施例中产率计算公式为：产率=产品灰度数据/(产品灰度数据+底物灰度数据)。The grayscale data of the product and substrate are obtained by grayscale analysis of the Urea-PAGE gel electrophoresis result image. The yield calculation formula in this embodiment is: yield = product grayscale data/(product grayscale data + substrate grayscale data).

实施例2：Embodiment 2:

挑选反应活性较好的Ligase 25，使用退火后的底物片段进行酶催化连接反应，反应条件如下：在50μL反应器中依次加入反应缓冲液(50mM Tris-HCl，pH 7.5)、三磷酸腺苷(adenosine triphosphate，ATP)、MgCl2、二硫苏糖醇(dithiothreitol，DTT)和RNA连接酶，16℃反应16h。Ligase 25 with good reaction activity was selected, and the annealed substrate fragments were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl 2 , dithiothreitol (DTT) and RNA ligase were added in sequence into a 50 μL reactor, and the reaction was carried out at 16° C. for 16 h.

反应结束后80℃加热5min使蛋白失活，离心取上清。送检HPLC和LC-MS检测，其中Ligase 25催化产物的HPLC结果如图3所示，“产率以反应体系样品的HPLC数据中产物峰的粗估占比进行衡量，结果如表3所示。After the reaction, the protein was inactivated by heating at 80°C for 5 min, and the supernatant was collected by centrifugation. The samples were sent for HPLC and LC-MS detection, and the HPLC results of the product catalyzed by Ligase 25 are shown in Figure 3. The yield was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample, and the results are shown in Table 3.

表3Table 3

。 .

备注：Remark:

1）反应条件：底物片段800μM，ATP 4eq，MgCl2 100eq，DTT 10eq（1eq=800μM），终浓度为0.2mg/mL酶，298V、50mM Tris-HCl pH 7.5，16℃，16h；1) Reaction conditions: substrate fragment 800 μM, ATP 4 eq, MgCl 2 100 eq, DTT 10 eq (1 eq = 800 μM), final concentration 0.2 mg/mL enzyme, 298 V, 50 mM Tris-HCl pH 7.5, 16 °C, 16 h;

2）+表示＜50%。2) + means <50%.

用LC-MS鉴定正义链产品分子量为6089.9，反义链产品分子量为6223.9，正义链产品理论值为6089.96±8，反义链产品理论值为6224.0±8，表明Ligase 25连接生成了QPI-1002，检测结果图如图4所示。The molecular weight of the sense chain product was identified by LC-MS to be 6089.9, and the molecular weight of the antisense chain product was 6223.9. The theoretical values of the sense chain product were 6089.96±8, and the theoretical values of the antisense chain product were 6224.0±8, indicating that QPI-1002 was generated by Ligase 25 ligation. The detection result is shown in Figure 4.

实施例3Example 3

使用退火后的底物片段和Ligase 25进行酶催化连接反应，反应条件如下：在10mL反应器中依次加入反应缓冲液(50mM Tris-HCl，pH 7.5)、三磷酸腺苷(adenosinetriphosphate，ATP)、MgCl2、二硫苏糖醇(dithiothreitol，DTT)和RNA连接酶，16℃反应16h。过夜反应结束后，50℃加热15min灭活蛋白，离心取上清，使用Nano-Q柱纯化，NaCl梯度洗脱，膜包(截留分子量1kDa)脱盐，冻干机内冻干得干粉，计算收率68.73%，纯度(HPLC检测)：95.44%。The annealed substrate fragment and Ligase 25 were used for enzyme catalytic ligation reaction. The reaction conditions were as follows: reaction buffer (50mM Tris-HCl, pH 7.5), adenosinetriphosphate (ATP), MgCl 2 , dithiothreitol (DTT) and RNA ligase were added to a 10mL reactor in sequence, and the reaction was carried out at 16°C for 16 hours. After the overnight reaction, the protein was inactivated by heating at 50°C for 15 minutes, and the supernatant was obtained by centrifugation and purified using a Nano-Q column, NaCl gradient elution, membrane package (molecular weight cutoff 1kDa) for desalting, and freeze-dried in a freeze dryer to obtain dry powder. The calculated yield was 68.73%, and the purity (HPLC detection) was 95.44%.

实施例4Example 4

基于QPI-1002的序列设计如表4所示的4个单链RNA片段，长度单位为nt。Four single-stranded RNA fragments were designed based on the sequence of QPI-1002 as shown in Table 4, with the length unit being nt.

表4Table 4

。 .

其中，A、C、G或U后的m表示对该核糖核苷酸的2’甲氧基修饰。The m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide.

底物5的第2、4、6和8位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4, 6 and 8 of substrate 5 have 2' methoxy modifications.

底物6的第2、4、6、8和10位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4, 6, 8 and 10 of substrate 6 have 2' methoxy modifications.

底物7的第1、3和5位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1, 3 and 5 of substrate 7 have 2’ methoxy modifications.

底物8的第1、3、5、7、9、11和13位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1, 3, 5, 7, 9, 11 and 13 of substrate 8 have 2' methoxy modifications.

使用固相合成方法制备上述4个单链RNA片段。The above four single-stranded RNA fragments were prepared using a solid phase synthesis method.

制备获得的QPI-1002的正义链为GAmGAmAUmAUmUUmCAmCCmCUmUCmA（SEQ ID NO：23）；反义链为UmGAmAGmGGmUGmAAmAUmAUmUCmUCm（SEQ ID NO：24）。The sense strand of the prepared QPI-1002 is GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23); the antisense strand is UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).

使用退火后的底物片段和连接酶Ligase 25进行酶催化连接反应，反应条件如下：将反应体系设置为50μL，在反应器中依次加入底物片段800μM，ATP 4eq，MgCl2 12.5eq，DTT1.25eq（1eq=800μM），终浓度为0.2mg/mL的Ligase 25，239V 50mM Tris-HCl pH 7.5，于16℃反应16 h。反应结束后80℃加热5min使蛋白失活，离心取上清。送检HPLC检测，产率以反应体系样品的HPLC数据中产物峰的粗估占比进行衡量。结果显示样品中目标峰占比为72.3%，即产率为+++。The annealed substrate fragment and ligase Ligase 25 were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: the reaction system was set to 50 μL, and 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl 2 , 1.25 eq DTT (1 eq = 800 μM) were added to the reactor in sequence, and Ligase 25 with a final concentration of 0.2 mg/mL was added, 239V 50 mM Tris-HCl pH 7.5, and reacted at 16 ° C for 16 h. After the reaction, the protein was inactivated by heating at 80 ° C for 5 min, and the supernatant was centrifuged. The sample was sent for HPLC detection, and the yield was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the target peak accounted for 72.3% in the sample, that is, the yield was +++.

实施例5Example 5

基于QPI-1002的序列设计如表5所示的6个单链RNA片段，长度单位为nt。Six single-stranded RNA fragments were designed based on the sequence of QPI-1002 as shown in Table 5, and the length unit is nt.

表5Table 5

。 .

其中，A、C、G或U后的m表示对该核糖核苷酸的2’甲氧基修饰。The m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide.

底物9的第2、4和6位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4 and 6 of substrate 9 have 2' methoxy modifications.

底物10的第2、4和6位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4 and 6 of substrate 10 have 2' methoxy modifications.

底物11的第1、3和5位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1, 3 and 5 of substrate 11 have 2' methoxy modifications.

底物12的第1和3位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1 and 3 of substrate 12 have 2' methoxy modifications.

底物13的第2、4和6位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 2, 4 and 6 of substrate 13 have 2' methoxy modifications.

底物14的第1、3、5、7和9位核糖核苷酸具有2’甲氧基修饰。The ribonucleotides at positions 1, 3, 5, 7 and 9 of substrate 14 have 2' methoxy modifications.

使用固相合成方法制备上述6个单链RNA片段。The above six single-stranded RNA fragments were prepared using a solid phase synthesis method.

制备获得的QPI-1002的正义链为GAmGAmAUmAUmUUmCAmCCmCUmUCmA（SEQ ID NO：23）；反义链为UmGAmAGmGGmUGmAAmAUmAUmUCmUCm（SEQ ID NO：24）。The sense strand of the prepared QPI-1002 is GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23); the antisense strand is UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).

使用退火后的底物片段和连接酶Ligase 25进行酶催化连接反应，反应条件如下：将反应体系设置为50μL，在反应器中依次加入底物片段800μM，ATP 4eq，MgCl2 12.5eq，DTT1.25eq（1eq=800μM），终浓度为0.2mg/mL的LigSase 25，239V 50mM Tris-HCl pH 7.5，于16℃反应16 h。反应结束后80℃加热5min使蛋白失活，离心取上清。送检HPLC检测，产率以反应体系样品的HPLC数据中产物峰的粗估占比进行衡量。结果显示样品中目标峰占比为79.6%，即产率为+++。The annealed substrate fragment and ligase Ligase 25 were used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: the reaction system was set to 50 μL, and 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl 2 , 1.25 eq DTT (1 eq = 800 μM) were added to the reactor in sequence, and LigSase 25 with a final concentration of 0.2 mg/mL, 239V 50 mM Tris-HCl pH 7.5, was reacted at 16 ° C for 16 h. After the reaction, the protein was inactivated by heating at 80 ° C for 5 min, and the supernatant was taken by centrifugation. The sample was sent for HPLC detection, and the yield was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the target peak accounted for 79.6% in the sample, that is, the yield was +++.

对比例1Comparative Example 1

利用固相合成全长QPI-1002产品的平均收率为29.5%，N+1和N-1杂质总占比为1.50%。The average yield of full-length QPI-1002 product using solid phase synthesis was 29.5%, and the total proportion of N+1 and N-1 impurities was 1.50%.

利用本发明中的酶连法制备获得的QPI-1002产品收率为66.18%，所用底物的固相合成收率平均值为45.9%，相乘可得整体流程的收率为30.3%，高于固相合成法中获得的产品平均收率，且N+1和N-1杂质总占比为0.38%低于固相合成法合成QPI-1002的过程该种杂质占比。The yield of the QPI-1002 product prepared by the enzyme-linked method of the present invention is 66.18%, and the average solid-phase synthesis yield of the substrate used is 45.9%. The yield of the overall process is 30.3%, which is higher than the average yield of the product obtained by the solid-phase synthesis method, and the total proportion of N+1 and N-1 impurities is 0.38%, which is lower than the proportion of such impurities in the process of synthesizing QPI-1002 by solid-phase synthesis.

从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：在本申请的制备方法中，首先合成4个短底物片段(长度小于19nt)，之后经酶连接合成全长的QPI-1002，从而实现利用生物合成的方式制备此种siRNA药物，由于缩短了合成片段的长度，合成过程产生的N+1和N-1杂质也相应减少；底物片段中的N+1和N-1杂质的连接效率降低，进一步降低了全长产品中的N+1和N-1杂质；此外，底物片段中的N+1和N-1杂质的链长同全长产品差异较大，易于在纯化过程中去除，最终N+1和N-1杂质的含量<0.5%。与化学合成制备法相比，本申请的制备方法获得的产物纯度高，产生的杂质少，制备过程简易，且反应条件温和，有机试剂用量低，降低生产成本，便于实现规模放大的工业化生产。From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the preparation method of the present application, four short substrate fragments (less than 19 nt in length) are first synthesized, and then the full-length QPI-1002 is synthesized by enzyme ligation, so as to realize the preparation of such siRNA drugs by biosynthesis. Since the length of the synthetic fragment is shortened, the N+1 and N-1 impurities generated in the synthesis process are also reduced accordingly; the connection efficiency of the N+1 and N-1 impurities in the substrate fragment is reduced, and the N+1 and N-1 impurities in the full-length product are further reduced; in addition, the chain length of the N+1 and N-1 impurities in the substrate fragment is quite different from that of the full-length product, and it is easy to remove them during the purification process, and the content of the final N+1 and N-1 impurities is <0.5%. Compared with the chemical synthesis preparation method, the product obtained by the preparation method of the present application has high purity, less impurities, simple preparation process, mild reaction conditions, low amount of organic reagents, reduced production costs, and is convenient for large-scale industrial production.

以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (13)

1. A method of preparing QPI-1002, wherein QPI-1002 is a double-stranded RNA consisting of a complementarily paired sense strand and antisense strand;
The preparation method comprises the following steps:
Mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the sense strand substrate fragment is capable of constituting the sense strand and the antisense strand substrate fragment is capable of constituting the antisense strand;
The sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, and the head base and the tail base of the sense strand substrate fragment and the antisense strand substrate fragment are not connected with each other to form a double-stranded nucleotide structure containing a nick;
connecting bases at two ends of the nick with a phosphodiester bond by using the RNA ligase to form the QPI-1002;
the bases at the two ends of the notch are respectively the 5 'end and the 3' end of different substrate fragments, the 5 'end is phosphate radical, and the 3' end is hydroxyl radical;
connecting the phosphate at the 5 'end and the hydroxyl at the 3' end at the upstream and downstream of the notch by using the RNA ligase to form the phosphodiester bond, so as to obtain the QPI-1002;
The RNA ligase comprises RNA ligase of RNA ligase family 1 and/or RNA ligase family 2;
The RNA ligase of the RNA ligase family 1 is SEQ ID NO:5, an RNA ligase shown in FIG. 5;
The RNA ligase of RNA ligase family 2 is selected from: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, one or more of the RNA ligases shown in fig. 4;
or with SEQ ID NO: 1-SEQ ID NO:5, and has an enzyme that catalyzes the formation of said phosphodiester bond.
2. The method of claim 1, wherein the nucleotide sequence of the sense strand is a sequence having the sequence of SEQ ID NO:23, and the nucleotide sequence of the antisense strand is a polynucleotide having the nucleotide sequence shown in SEQ ID NO:24, and a polynucleotide having a nucleotide sequence shown in seq id no.
3. The method of any one of claims 1-2, wherein the sense strand substrate fragment comprises 2 or more and the antisense strand substrate fragment comprises 2 or more;
the length of the sense strand substrate fragment is 5-12nt;
the length of the antisense strand substrate fragment is 2-16nt.
4. The method of preparing according to claim 3, wherein the sense strand substrate fragment and the antisense strand substrate fragment each comprise 2, the sense strand substrate fragment comprises a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragment comprises a first antisense strand substrate fragment and a second antisense strand substrate fragment;
The preparation method comprises the following steps:
S1) mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the RNA ligase;
S2) under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form the sense strand, the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to be connected to form the antisense strand, and the sense strand and the antisense strand are formed into the QPI-1002 through base complementation pairing;
Annealing the first sense strand substrate fragment and the first antisense strand substrate fragment to form a first double-stranded RNA fragment, and annealing the second sense strand substrate fragment and the second antisense strand substrate fragment to form a second double-stranded RNA fragment, wherein base complementary pairing is more than or equal to 3nt in each of the first double-stranded RNA fragment and the second double-stranded RNA fragment;
And a sticky end can be formed between the first double-stranded RNA fragment and the second double-stranded RNA fragment, and the length of the sticky end is more than or equal to 2nt.
5. The method of claim 4, wherein in S1) the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:9, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:10, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:12, and the nucleotide sequence of the second antisense strand substrate fragment is set forth in SEQ ID NO:11, and a nucleotide sequence shown in the sequence No. 11.
6. The method of claim 4, wherein in S1), the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:13, and the nucleotide sequence of the second sense strand substrate fragment is set forth in SEQ ID NO:14, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:16, and the nucleotide sequence of the second antisense strand substrate fragment is set forth in SEQ ID NO:15, and a nucleotide sequence shown in seq id no.
7. The method of claim 4, wherein in S1), the 3 'end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are linked under the catalysis of the RNA ligase to form the sense strand; the 3 'end of the first antisense strand substrate fragment is ligated to the 5' end of the second antisense strand substrate fragment under the catalysis of the RNA ligase to form the antisense strand.
8. The method of claim 7, wherein the first sense strand substrate fragment has a hydroxyl group at the 5 'end and a hydroxyl group at the 3' end; the 5 'end of the second sense strand substrate fragment is a phosphate group, and the 3' end of the second sense strand substrate fragment is a hydroxyl group;
the 5 'end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end of the first antisense strand substrate fragment is a hydroxyl group; the 5 'end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.
9. The method according to claim 3, wherein the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3,
The sense strand substrate fragments comprise a first sense strand substrate fragment, a second sense strand substrate fragment, and a third sense strand substrate fragment;
The antisense strand substrate fragments include a first antisense strand substrate fragment, a second antisense strand substrate fragment, and a third antisense strand substrate fragment.
10. The method of preparing according to claim 9, wherein the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3 pieces, the method of preparing comprising: s1) mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment and the RNA ligase;
S2) under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form the sense strand, the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to be connected to form the antisense strand, and the sense strand and the antisense strand are formed into the QPI-1002 through base complementation pairing.
11. The method of claim 10, wherein in S1) the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:17, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:18, a nucleotide sequence shown in seq id no;
the nucleotide sequence of the third sense strand substrate fragment is SEQ ID NO:19, a nucleotide sequence shown in seq id no;
The first antisense strand substrate fragment is SEQ ID NO:20, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO:21, a nucleotide sequence shown in seq id no;
the nucleotide sequence of the third antisense strand substrate fragment is SEQ ID NO:22, and a nucleotide sequence shown in seq id no.
12. The method of any one of claims 1-2, wherein the sense strand substrate fragment and the antisense strand substrate fragment concentrations are each independently selected from 0.1-4.5mM;
The reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase further comprises ATP, tris-HCl, mgCl 2 and DTT.
13. The preparation method according to claim 1, wherein the reaction temperature of the preparation method is 0 ℃ to 60 ℃;
the reaction time of the preparation method is 0.5-24 hours;
The pH of the preparation method is 6-8.5.
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