[go: up one dir, main page]

CN118460648B - A preparation method of QPI-1002 - Google Patents

A preparation method of QPI-1002 Download PDF

Info

Publication number
CN118460648B
CN118460648B CN202410921728.1A CN202410921728A CN118460648B CN 118460648 B CN118460648 B CN 118460648B CN 202410921728 A CN202410921728 A CN 202410921728A CN 118460648 B CN118460648 B CN 118460648B
Authority
CN
China
Prior art keywords
substrate fragment
sense strand
antisense strand
strand substrate
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202410921728.1A
Other languages
Chinese (zh)
Other versions
CN118460648A (en
Inventor
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
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Kailaiying Biotechnology Co ltd
Asymchem Laboratories Tianjin Co Ltd
Original Assignee
Tianjin Kailaiying Biotechnology Co ltd
Asymchem Laboratories Tianjin Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Kailaiying Biotechnology Co ltd, Asymchem Laboratories Tianjin Co Ltd filed Critical Tianjin Kailaiying Biotechnology Co ltd
Priority to CN202410921728.1A priority Critical patent/CN118460648B/en
Publication of CN118460648A publication Critical patent/CN118460648A/en
Application granted granted Critical
Publication of CN118460648B publication Critical patent/CN118460648B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明提供了一种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。能够解决现有技术中制备QPI‑1002纯度较低的问题，适用于药物生物合成技术领域。

The present invention provides a preparation method of QPI-1002. The 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, forming a double-stranded nucleotide structure containing a nick; and the bases at both ends of the nick are connected by a phosphodiester bond using an RNA ligase to form QPI-1002. The method can solve the problem of low purity of QPI-1002 prepared in the prior art, and is applicable to the field of drug biosynthesis technology.

Description

Preparation method of QPI-1002
Technical Field
The invention relates to the field of medicine biosynthesis, in particular to a preparation method of QPI-1002.
Background
The small interfering RNA (SMALL INTERFERING RNA, SIRNA) is double-stranded RNA with the length of 19-25nt, the siRNA can be dissociated into single strands after entering cells, wherein the sense strand can be specifically combined with messenger RNA (MESSENGER RNA, MRNA) of a target gene through base matching, a series of effects are induced, mRNA of the target gene is finally degraded, and translation of the mRNA is prevented so as to achieve the effect of obstructing target gene expression. In recent years, the development of siRNA drugs has achieved extensive attention, the siRNA drugs act on mRNA, and the target point of the ready-made medicine is obviously larger than that of the traditional small molecular medicine with the acting site being protein; in addition, siRNA drugs can act on new targets by transforming sequences, and development time is relatively short.
QPI-1002 is siRNA double-stranded RNA drug developed by Quark corporation, and is currently in clinical phase 3 experimental stage. QPI-1002 is useful for kidney related diseases, such as inhibiting Major renal adverse events (Major ADVERSE KIDNEY EVENTS, MAKE).
Currently, QPI-1002 is prepared by chemical methods, using solid phase carriers such as controllable microporous glass beads (Controlled Pore Glass, CPG) or polystyrene resin, circularly synthesizing by phosphoramidite triester method, extending oligonucleotide chain along 3 'to 5', cutting QPI-1002 chain from the solid phase carrier by ammonolysis after synthesis cycle is completed, and purifying to obtain pure product. Solid phase synthesis is a cyclic method, the yield of synthesis decreases with increasing synthetic chain length, and impurities generated in the synthesis process, such as impurities (n+1 impurities) one nucleotide more than the target sequence or impurities (N-1 impurities) one nucleotide less than the target sequence, also increase with increasing synthetic chain length, such impurities are not easily removed, resulting in complicated purification process and low efficiency. Thus QPI-1002 is expensive to prepare and difficult to scale up, and thus a more efficient QPI-1002 synthesis method needs to be developed.
Disclosure of Invention
The invention mainly aims to provide a preparation method of QPI-1002, which aims to solve the problem of low purity of QPI-1002 prepared in the prior art.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a method for preparing QPI-1002, which QPI-1002 is a double stranded RNA composed of complementarily paired sense strand and antisense strand; the preparation method comprises the steps of mixing a sense strand substrate fragment, an antisense strand substrate fragment and RNA ligase, wherein the sense strand substrate fragment can form a sense strand, the antisense strand substrate fragment can form an antisense strand, the sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, 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 notch, the bases at the two ends of the notch are connected through a phosphodiester bond by utilizing the RNA ligase to form QPI-1002, the bases at the two ends of the notch are respectively a5 'end and a 3' end of different substrate fragments, the 5 'end is a phosphate radical, the 3' end is a hydroxyl radical, the RNA ligase is utilized to connect the phosphate radical at the 5 'end at the upstream and the downstream of the notch and the hydroxyl at the 3' end of the antisense strand substrate fragment, the RNA ligase comprises RNA ligase family 1 and/or RNA ligase of RNA ligase family 2, preferably, the RNA ligase of the RNA ligase family 1 is RNA ligase of the RNA ligase, the RNA ligase has the RNA ligase of the RNA ligase family 1, the RNA ligase of the RNA ligase 1 is the RNA ligase of the SEQ ID 2, and the RNA ligase has the RNA ligase 1 or the RNA ligase 1 shown in the SEQ 1-5-3-5-SEQ 1.
Further, 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.
Further, the sense strand substrate fragment comprises 2 or more, the antisense strand substrate fragment comprises 2 or more, preferably the sense strand substrate fragment has a length of 5 to 12nt, more preferably 6 to 11nt, and preferably the antisense strand substrate fragment has a length of 2 to 16nt, more preferably 3 to 14nt.
Further, 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 steps of S1) mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a first antisense strand substrate fragment, a second antisense strand substrate fragment and RNA ligase, S2) connecting the first sense strand substrate fragment and the second sense strand substrate fragment under the catalysis of the RNA ligase to form a sense strand, catalyzing the first antisense strand substrate fragment and the second antisense strand substrate fragment to connect to form an antisense strand, and forming QPI-1002 by base complementation of the sense strand and the antisense strand, wherein the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with the RNA ligase to obtain QPI-1002, the first sense strand substrate fragment and the first antisense strand substrate fragment are annealed to form a first double-stranded RNA fragment, the second sense strand substrate fragment and the second antisense strand substrate fragment are annealed to form a second double-stranded RNA fragment, base complementation of not less than 3 is catalyzed in the first double-stranded RNA fragment and the second double-stranded RNA fragment, and the cohesive end between the first double-stranded RNA fragment and the second double-stranded RNA fragment is not less than 2.
Further in S1), the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 9, the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 10, preferably the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 12, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 11.
Further in S1), the nucleotide sequence of the first sense strand substrate fragment is shown as SEQ ID NO. 13, the nucleotide sequence of the second sense strand substrate fragment is shown as SEQ ID NO. 14, preferably the first antisense strand substrate is shown as SEQ ID NO. 16, and the second antisense strand substrate is shown as SEQ ID NO. 15.
Further in S1), the 3' end of the first sense strand substrate fragment is connected with the 5' end of the second sense strand substrate fragment under the catalysis of RNA ligase to form a sense strand, the 3' end of the first antisense strand substrate fragment is connected with the 5' end of the second antisense strand substrate fragment under the catalysis of RNA ligase to form an antisense strand, preferably, the 5' end of the first sense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, the 5' end of the second sense strand substrate fragment is a phosphate group, the 3' end is a hydroxyl group, preferably, the 5' 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.
Further, the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3, the sense strand substrate fragment comprises a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment; the antisense strand substrate fragments comprise 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 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 a sense strand, the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to form an antisense strand, the sense strand and the antisense strand are formed into QPI-1002 through base complementation pairing, preferably the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with the RNA ligase to obtain QPI-1002, preferably the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:17, the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:17, the nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19), the nucleotide sequence of the first antisense strand substrate fragment is shown as SEQ ID NO. 20, the nucleotide sequence of the second antisense strand substrate fragment is shown as SEQ ID NO. 21, and the nucleotide sequence of the third antisense strand substrate fragment is shown as SEQ ID NO. 22.
Further, the concentration of the sense strand substrate fragment and the antisense strand substrate fragment are each independently selected from 0.1-4.5mM, preferably 0.8-1.6mM, 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 0-60 ℃, more preferably 4-37 ℃, preferably, the reaction time of the preparation method is 0.5-24 h, more preferably 12-16h, and the pH of the preparation method is 6-8.5.
By applying the technical scheme of the application, the preparation method is utilized, under the catalysis of RNA ligase, the sense strand substrate fragments are connected to form QPI-1002 sense strand, and the antisense strand substrate fragments are connected to form QPI-1002 antisense strand, so that the siRNA medicine is prepared by utilizing a biosynthesis mode. Compared with the method for preparing QPI-1002 by chemical synthesis, the preparation method provided by the application has the advantages that the purity of the obtained product is high, the generated impurities are less, the preparation process is simple, the reaction condition is mild, the dosage of organic reagent is low, the production cost is reduced, and the large-scale industrialized production is convenient to realize.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows a schematic of an enzyme-catalyzed ligation reaction according to example 1 of the invention.
FIG. 2 shows a graph of the electrophoresis results of catalytic products of RNA Ligase Ligase 31, ligase 25 and Ligase 11 according to example 1 of the present invention.
FIG. 3 shows a graph of HPLC detection of the RNA Ligase Ligase 25 catalytic product according to example 2 of the present invention.
FIG. 4 shows a graph of LC-MS detection results of the catalytic product of RNA Ligase 25 according to example 2 of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
Term interpretation:
N+1 impurities nucleic acid impurities having a single nucleotide attached in addition to the target synthetic sequence.
N-1 impurities nucleic acid impurities having a single nucleotide deletion compared to the target synthetic sequence.
As mentioned in the background art, the preparation of QPI-1002 in the prior art is carried out by adopting a chemical synthesis preparation method, so that the process is complex, the cost is high, and the generated N+1 and N-1 impurities are more, thereby influencing the subsequent purification of the product. In the present application, the inventors have attempted to develop a method for preparing QPI-1002 by preparing QPI-1002 using enzymatic synthesis, and thus have proposed a series of protection schemes of the present application.
In a first exemplary embodiment of the application, a method of preparing QPI-1002 is provided, the QPI-1002 being a double stranded RNA consisting of complementarily paired sense and antisense strands; the preparation method comprises the steps of mixing a sense strand substrate fragment, an antisense strand substrate fragment and RNA ligase, wherein the sense strand substrate fragment can form a sense strand, the antisense strand substrate fragment can form an antisense strand, the sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, 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 notch, the bases at the two ends of the notch are connected through a phosphodiester bond by utilizing the RNA ligase to form QPI-1002, the bases at the two ends of the notch are respectively a 5 'end and a 3' end of different substrate fragments, the 5 'end is a phosphate radical, the 3' end is a hydroxyl radical, the RNA ligase is utilized to connect the phosphate radical at the 5 'end at the upstream and the downstream of the notch and the hydroxyl at the 3' end of the antisense strand substrate fragment, the RNA ligase comprises RNA ligase family 1 and/or RNA ligase of RNA ligase family 2, preferably, the RNA ligase of the RNA ligase family 1 is RNA ligase of the RNA ligase, the RNA ligase has the RNA ligase of the RNA ligase family 1, the RNA ligase of the RNA ligase 1 is the RNA ligase of the SEQ ID 2, and the RNA ligase has the RNA ligase 1 or the RNA ligase 1 shown in the SEQ 1-5-3-5-SEQ 1.
In the above preparation method, the sense strand substrate fragment is a nucleotide sequence of 2 or more that can constitute the sense strand, i.e., a plurality of nucleotide sequences of the sense strand substrate fragment can be spliced to constitute the same sequence as the sense strand sequence, and is different from the sense strand in that there is a nick between the sense strand substrate fragments, which are not linked by a phosphodiester bond. Similarly, the antisense strand substrate fragment and antisense strand have the features described above. The sense strand and the antisense strand of QPI-1002 are obtained by connecting 2 or more sense strand substrate fragments or antisense strand substrate fragments with each other by phosphodiester bonds by RNA ligase.
In the above preparation method, QPI-1002 can be prepared by mixing the sense strand substrate fragment and the antisense strand substrate fragment with RNA ligase. In the preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be complementarily paired to form a double-stranded structure nucleotide containing a sticky end, the double-stranded structure nucleotide containing the sticky end is continuously combined with other substrate fragments to form a double-stranded nucleotide containing an nick, and the RNA ligase can recognize the nick of the double-stranded structure and connect the nick by a phosphodiester bond, so that the target product QPI-1002 is prepared.
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:
GAmGAmAUmAUmUUmCAmCCmCUmUCmA。
SEQ ID NO:24:
UmGAmAGmGGmUGmAAmAUmAUmUCmUCm。
In the present application, A, C, G or m after U represents 2' methoxy modification of the ribonucleotide.
In a preferred embodiment, the RNA ligase comprises RNA ligase 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, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98.5%, 99.5%, 99.6%, 99.7%, 99.8% or more, even 99.9% or more) and having catalytic phosphodiester bond formation activity with any of the RNA ligases shown in SEQ ID NO: 1-SEQ ID NO: 5.
The RNA ligase can recognize a double-chain structure with a notch formed by complementary pairing of substrate fragments, so that the formation of a phosphodiester bond between a phosphate group and a hydroxyl group is catalyzed.
SEQ ID NO:1(Ligase 25,Vibrio phage NT-1):
MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.
SEQ ID NO:2(Ligase 26,Escherichia phage AR1):
MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY.
SEQ ID NO:3(Ligase 31,Vibrio phage VH12019):
MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAHADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA.
SEQ ID NO:4:(Ligase 41,Vibrio phage VH7D):
MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATAHEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV.
SEQ ID NO:5(Ligase 42,Escherichia phage JN02):
MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY.
SEQ ID NO:6:(Ligase 11,Thermococcus):
MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.
SEQ ID NO:7:(Ligase 20,Archaea):
MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.
SEQ ID NO:8:(Ligase 32,Bacteria):
MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.
Identity (Identity) in the present application refers to "Identity" between amino acid sequences or nucleotide sequences, i.e. the sum of the ratios of amino acid residues or nucleotides of the same kind in an amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using the alignment programs BLAST (Basic Local ALIGNMENT SEARCH Tool), FASTA, etc.
Proteins that are 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., 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) identical and have the same function have the same active site, active pocket, active mechanism, protein structure, etc. as those provided by the above sequences with a high probability.
As used herein, amino acid residues are abbreviated as 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).
The rules of substitution, replacement, etc., generally, which amino acids are similar in nature, and the effect after replacement is similar. For example, in the above homologous proteins, conservative amino acid substitutions may occur. "conservative amino acid substitutions" include, but are not limited to:
The hydrophobic amino acid (Ala, cys, gly, pro, met, val, ile, leu) is substituted with other hydrophobic amino acids;
The hydrophobic amino acid (Phe, tyr, trp) with a coarse side chain is replaced by other hydrophobic amino acids with a coarse side chain;
The positively charged amino acid (Arg, his, lys) of the side chain is replaced by other positively charged amino acids of the side chain;
The amino acid (Ser, thr, asn, gln) with a side chain having a polarity that is uncharged is substituted with other amino acids with a side chain having a polarity that is uncharged.
The amino acids may also be conservatively substituted by those skilled in the art according to amino acid substitution rules well known to those skilled in the art as the "blosum62 scoring matrix" in the art.
In the application, the phosphodiester bond is formed by the phosphate group and the hydroxyl group of the substrate only through the RNA ligase shown in SEQ ID NO. 1-SEQ ID NO. 5 or the enzyme which has more than 70% of the same degree with any RNA ligase shown in SEQ ID NO. 1-SEQ ID NO. 5, so that the product QPI-1002 is obtained. In the related experiments of the application, the inventors obtained RNA ligase shown by SEQ ID NO. 1-SEQ ID NO. 5 capable of synthesizing QPI-1002 by screening from a large number of enzymes. A negative result with a large proportion in the experiment shows that most RNA ligases are difficult to catalyze the synthesis of QPI-1002, including but not limited to the RNA ligases shown in SEQ ID NO. 6-SEQ ID NO. 8, and the RNA ligases without activity of catalyzing the synthesis of QPI-1002 are only shown by taking SEQ ID NO. 6-SEQ ID NO. 8 as an example in the specification of the application.
In a preferred embodiment, the sense strand substrate fragment comprises 2 or more, the antisense strand substrate fragment comprises 2 or more, preferably the sense strand substrate fragment has a length of 5 to 12nt, more preferably 6 to 11nt, preferably the antisense strand substrate fragment has a length of 2 to 16nt, more preferably 3 to 14nt.
In a preferred embodiment, 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 steps of S1) mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a first antisense strand substrate fragment and a second antisense strand substrate fragment, S2) connecting the first sense strand substrate fragment and the second sense strand substrate fragment under the catalysis of RNA ligase to form a sense strand, catalyzing the first antisense strand substrate fragment and the second antisense strand substrate fragment to connect to form an antisense strand, and forming QPI-1002 by complementary base pairing of the sense strand and the antisense strand, wherein the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain QPI-1002, the first sense strand substrate fragment and the first antisense strand substrate fragment are annealed to form a first double-stranded RNA fragment, the second sense strand substrate fragment and the second antisense strand substrate fragment are annealed to form a second double-stranded RNA fragment, and complementary pairing of 3nt or more is formed in the first double-stranded RNA fragment and the second double-stranded RNA fragment, and the cohesive end between the first double-stranded RNA fragment and the second double-stranded RNA fragment can form a cohesive end or more than or equal to 2nt.
In the preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be mixed for annealing, and a double-stranded RNA structure can be formed by base complementary pairing between the sense strand substrate fragment and the antisense strand substrate fragment, and the double-stranded RNA structure has a notch between different substrate fragments. And mixing the annealed reaction system with RNA ligase, connecting phosphate groups and hydroxyl groups at two sides of the notch by using the RNA ligase through a phosphodiester bond, and repairing the notch, thereby obtaining a target product QPI-1002 with complete double-chain structure.
In a preferred embodiment, 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 as SEQ ID NO. 12 and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown as SEQ ID NO. 11.
In a preferred embodiment, 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 as SEQ ID NO. 16 and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown as SEQ ID NO. 15.
In a preferred embodiment, S2), the 3 'end of the first sense strand substrate fragment is linked to the 5' end of the second sense strand substrate fragment under the catalysis of RNA ligase to form a sense strand, the 3 'end of the first antisense strand substrate fragment is linked to the 5' end of the second antisense strand substrate fragment under the catalysis of RNA ligase to form an antisense strand, preferably the 5 'end of the first sense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, the 5 'end of the second sense strand substrate fragment is a phosphate group, the 3' end is a hydroxyl group, preferably the 5 'end of the first antisense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, and the 5 'end of the second antisense strand substrate fragment is a phosphate group, the 3' end is a hydroxyl group.
QPI-1002 can be prepared using the preparation method described above and the substrate fragment shown as 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 the substrate fragment is not limited to the substrate fragments shown in SEQ ID NOS.9-12 or SEQ ID NOS.13-16, and the substrate fragments capable of being combined to form the sense strand and the antisense strand can be applied to the preparation method described above, which is applicable to the preparation of QPI-1002, but not limited to the difference in the ligation positions of the substrate fragments, and the preparation described above has a good ligation effect for the ligation of the sense strand sequence and the antisense strand sequence of QPI-1002. The number of sense strand substrate fragments or antisense strand substrate fragments includes, but is not limited to, 2,3, 4, or even more.
SEQ ID NO:9:GAmGAmAUmAUmUUmCAmC。
SEQ ID NO:10:CmCUmUCmA。
SEQ ID NO:11:AUmAUmUCmUCm。
SEQ ID NO:12:UmGAmAGmGGmUGmAAm。
SEQ ID NO:13:GAmGAmAUmAUm。
SEQ ID NO:14:UUmCAmCCmCUmUCmA。
SEQ ID NO:15:UmUCmUCm。
SEQ ID NO:16:UmGAmAGmGGmUGmAAmAUmA。
In a preferred embodiment, the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3, the sense strand substrate fragment comprising a first sense strand substrate fragment, a second sense strand substrate fragment, and a third sense strand substrate fragment; the antisense strand substrate fragments comprise 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 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 a sense strand, the first antisense strand substrate fragment and the second antisense strand substrate fragment are catalyzed to form an antisense strand, the sense strand and the antisense strand are formed into QPI-1002 through base complementation pairing, preferably the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with the RNA ligase to obtain QPI-1002, preferably the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:17, the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO:17, the nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19), the nucleotide sequence of the first antisense strand substrate fragment is shown as SEQ ID NO. 22, the nucleotide sequence of the second antisense strand substrate fragment is shown as SEQ ID NO. 21, and the nucleotide sequence of the third antisense strand substrate fragment is shown as SEQ ID NO. 20.
SEQ ID NO:17:GAmGAmAUm。
SEQ ID NO:18:AUmUUmCAmC。
SEQ ID NO:19:CmCUmUCmA。
SEQ ID NO:20:CmUCm。
SEQ ID NO:21:AAmAUmAUmU。
SEQ ID NO:22:UmGAmAGmGGmUGm。
In a preferred embodiment, the concentration of the sense strand substrate fragment and the antisense strand substrate fragment is respectively selected from 0.1-4.5mM, preferably 0.8-1.6mM, 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 0-60 ℃, more preferably 4-37 ℃, preferably, the reaction time of the preparation method is 0.5-24 h, more preferably 12-16h, and the pH of the preparation method is 6-8.5.
The concentration of the sense strand substrate fragment and the antisense strand substrate fragment is selected from the group consisting of, 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 4.5mM, the reaction temperature of the preparation method includes, but not limited to, 10, 15, 16, 20, 25, 30, 35, or 40 ℃, the reaction time of the preparation method includes, but not limited to, 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, or 48 hours, and the pH of the preparation method includes, but not limited to, 6, 6.5, 7, 7.5, 8, or 8.5.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1
The 4 single stranded RNA fragments shown in Table 1 were designed based on the QPI-1002 sequence in nt length units.
TABLE 1
。
Wherein A, C, G or m after U represents 2' methoxy modification of the ribonucleotide.
Ribonucleotides at positions 2, 4, 6, 8, 10 and 12 of substrate 1 have 2' methoxy modifications.
Ribonucleotides at positions 1, 3 and 5 of substrate 2 have 2' methoxy modifications.
Ribonucleotides at positions 2, 4, 6 and 8 of substrate 3 have 2' methoxy modifications.
Ribonucleotides at positions 1, 3,5, 7, 9 and 11 of substrate 4 have 2' methoxy modifications.
The above 4 single-stranded RNA fragments were prepared using a solid phase synthesis method.
The 4 single-stranded RNA fragments were mixed in an equimolar ratio to obtain a substrate mixture having a final concentration of 2.5mM (2.5 mM for each substrate), and annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to enzyme-catalyzed ligation reaction, the reaction system was set to 10. Mu.L, and the reaction system included reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (adenosine triphosphate, ATP), mgCl 2, dithiothreitol (DTT), and RNA Ligase Ligase 25, ligase 26, ligase 31, ligase 41, ligase 42, ligase 11, ligase 20 and Ligase 32 were added, respectively. The reaction was reacted at 16 ℃ for 16h. The resulting reaction system was subjected to enzyme inactivation of ligase at 80℃for 5min and centrifugation at 12000rpm to remove precipitate. The enzyme-catalyzed ligation reaction is schematically depicted in FIG. 1.
SDS-PAGE detection was performed on products obtained by catalyzing with RNA ligases Ligase25, ligase 26, ligase31, ligase 41, ligase 42 Ligase11, ligase 20 and Ligase 32. The electrophoresis results of the products obtained by catalyzing the RNA Ligase31, ligase25 and Ligase11 are shown in FIG. 2, wherein the lane M in FIG. 2 represents the RNA molecular standard (marker), the lane 1 represents the reaction system of Ligase31, the lane 2 represents the reaction system of Ligase25, and the lane 3 represents the reaction system of Ligase 11. The yields were estimated from the gray scale analysis of the target bands in the Urea-PAGE results, and the final yield results are shown in Table 2.
The activity screening of 10 RNA ligases shows that the Ligase25 has better connection effect and can convert most substrates into QPI-1002.
The sense strand of QPI-1002 prepared was GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23) and the antisense strand was UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).
The reaction results are shown in Table 2.
TABLE 2
。
Remarks:
1) The reaction conditions were 100. Mu.M for the substrate fragment, 10eq for ATP, 2 eq for MgCl, 10eq for DTT (1 eq=100. Mu.M) for a final concentration of 0.2mg/mL enzyme, 2385V, 50 mM Tris-HCl pH 7.5,16 ℃for 16 h;
2) N.d. indicates that no product formation is detected, ++indicates 25-50% (excluding 50% of the end point values), ++ represents 50-75%, 50-75 percent (a).
The yield calculation formula in this example is yield = product gray data/(product gray data + substrate gray data).
Example 2:
Ligase 25 with better reactivity was selected and the annealed substrate fragment was used for enzyme-catalyzed ligation under the following conditions, reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (adenosine triphosphate, ATP), mgCl 2, dithiothreitol (DTT) and RNA Ligase were added sequentially in a 50. Mu.L reactor and reacted for 16h at 16 ℃.
Heating at 80deg.C for 5min to deactivate protein, and centrifuging to obtain supernatant. The HPLC and LC-MS measurements were performed, wherein the HPLC results of the Ligase 25 catalytic product are shown in FIG. 3, and the "yield was measured as the roughly estimated ratio of the product peak in the HPLC data of the reaction system sample, and the results are shown in Table 3.
TABLE 3 Table 3
。
Remarks:
1) The reaction conditions were 800. Mu.M substrate fragment, ATP 4eq, mgCl 2 eq, DTT 10eq (1 eq=800. Mu.M), final concentration of 0.2mg/mL enzyme, 298V, 50mM Tris-HCl pH 7.5,16 ℃for 16h;
2) + represents <50%.
LC-MS is used for identifying that the molecular weight of a sense strand product is 6089.9, the molecular weight of an antisense strand product is 6223.9, the theoretical value of the sense strand product is 6089.96 +/-8, and the theoretical value of the antisense strand product is 6224.0 +/-8, which shows that the ligation of Ligase 25 generates QPI-1002, and the detection result is shown in a graph in FIG. 4.
Example 3
The annealed substrate fragment and Ligase 25 were used for the enzyme-catalyzed ligation reaction under the following conditions of sequentially adding a reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (adenosine triphosphate, ATP), mgCl 2, dithiothreitol (DTT) and RNA Ligase in a 10mL reactor, and reacting at 16℃for 16 hours. After the overnight reaction, heating at 50deg.C for 15min to inactivate protein, centrifuging to obtain supernatant, purifying with Nano-Q column, gradient eluting with NaCl, desalting with membrane bag (molecular weight cut-off 1 kDa), lyophilizing in a lyophilizer to obtain dry powder, and calculating yield 68.73% and purity (HPLC detection) 95.44%.
Example 4
The 4 single stranded RNA fragments shown in Table 4 were designed based on the QPI-1002 sequence in nt length units.
TABLE 4 Table 4
。
Wherein A, C, G or m after U represents 2' methoxy modification of the ribonucleotide.
Ribonucleotides at positions 2, 4, 6 and 8 of substrate 5 have 2' methoxy modifications.
Ribonucleotides at positions 2, 4, 6, 8 and 10 of substrate 6 have 2' methoxy modifications.
Ribonucleotides at positions 1, 3 and 5 of substrate 7 have 2' methoxy modifications.
Ribonucleotides at positions 1,3, 5, 7, 9, 11 and 13 of substrate 8 have 2' methoxy modifications.
The above 4 single-stranded RNA fragments were prepared using a solid phase synthesis method.
The sense strand of QPI-1002 prepared was GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23) and the antisense strand was UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).
The enzyme-catalyzed ligation was performed using the annealed substrate fragment and Ligase Ligase 25 under the conditions in which the reaction system was set to 50. Mu.L, and 800. Mu.M substrate fragment, ATP 4eq, mgCl 2 12.5eq,DTT 1.25eq (1 eq=800. Mu.M) were sequentially added to the reactor, and Ligase 25,239V 50mM Tris-HCl pH 7.5 was reacted at 16℃and 16 h at a final concentration of 0.2 mg/mL. Heating at 80deg.C for 5min to deactivate protein, and centrifuging to obtain supernatant. The assay HPLC was run and the yield was measured as the coarsely estimated occupancy of the product peak in the HPLC data of the reaction system samples. The results showed a target peak ratio of 72.3% in the sample, i.e. a yield of++.
Example 5
The 6 single stranded RNA fragments shown in Table 5 were designed based on the QPI-1002 sequence in nt length units.
TABLE 5
。
Wherein A, C, G or m after U represents 2' methoxy modification of the ribonucleotide.
Ribonucleotides at positions 2, 4 and 6 of substrate 9 have 2' methoxy modifications.
Ribonucleotides at positions 2,4 and 6 of substrate 10 have 2' methoxy modifications.
Ribonucleotides at positions 1, 3 and 5 of substrate 11 have 2' methoxy modifications.
Ribonucleotides at positions 1 and 3 of substrate 12 have 2' methoxy modifications.
Ribonucleotides at positions 2,4 and 6 of substrate 13 have 2' methoxy modifications.
Ribonucleotides at positions 1, 3, 5, 7 and 9 of substrate 14 have 2' methoxy modifications.
The above 6 single-stranded RNA fragments were prepared using a solid phase synthesis method.
The sense strand of QPI-1002 prepared was GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23) and the antisense strand was UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).
The enzyme-catalyzed ligation was performed using the annealed substrate fragment and Ligase Ligase 25 under the conditions in which the reaction system was set to 50. Mu.L, 800. Mu.M substrate fragment, ATP 4eq, mgCl 2 12.5eq,DTT 1.25eq (1 eq=800. Mu.M) were added sequentially to the reactor, and LigSase 25,239V 50mM Tris-HCl pH 7.5 was reacted at 16℃to h at a final concentration of 0.2 mg/mL. Heating at 80deg.C for 5min to deactivate protein, and centrifuging to obtain supernatant. The assay HPLC was run and the yield was measured as the coarsely estimated occupancy of the product peak in the HPLC data of the reaction system samples. The results showed that the target peak ratio in the sample was 79.6%, i.e. the yield was++.
Comparative example 1
The average yield of the full length QPI-1002 product synthesized using the solid phase was 29.5% with a total of 1.50% N+1 and N-1 impurities.
The yield of QPI-1002 obtained by the enzyme-linked method is 66.18%, the average value of the solid phase synthesis yield of the used substrate is 45.9%, the yield of the multiplied integral process is 30.3%, the product is higher than the average yield of the product obtained by the solid phase synthesis method, and the total impurity ratio of N+1 and N-1 is 0.38% which is lower than the impurity ratio in the process of synthesizing QPI-1002 by the solid phase synthesis method.
From the above description, it can be seen that the above embodiment of the present application achieves the technical effects that in the preparation method of the present application, 4 short substrate fragments (length is less than 19 nt) are synthesized first, and then the full-length QPI-1002 is synthesized by enzyme ligation, thereby realizing the preparation of the siRNA drug by using the biosynthesis method, since the length of the synthesized fragments is shortened, the N+1 and N-1 impurities generated during the synthesis process are correspondingly reduced, the ligation efficiency of the N+1 and N-1 impurities in the substrate fragments is reduced, the N+1 and N-1 impurities in the full-length product are further reduced, and in addition, the chain length of the N+1 and N-1 impurities in the substrate fragments is greatly different from that of the full-length product, and is easy to remove during the purification process, and the content of the final N+1 and N-1 impurities is less than 0.5%. Compared with a chemical synthesis preparation method, the preparation method provided by the application has the advantages that the purity of the obtained product is high, the generated impurities are less, the preparation process is simple, the reaction condition is mild, the dosage of organic reagent is low, the production cost is reduced, and the large-scale industrial production is convenient to realize.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (11)

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;
a part of the sense strand substrate fragment and a part of the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation to form a double-stranded nucleotide structure with a sticky end;
The double-stranded nucleotide structure containing the sticky end is continuously connected with the rest of the sense strand substrate fragment and the rest of the antisense strand substrate fragment 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 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, wherein the 5 '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 is RNA ligase of RNA ligase family 1 and/or RNA ligase family 2;
The RNA ligase of the RNA ligase family 1 is the RNA ligase shown in SEQ ID NO. 5;
The RNA ligase of the RNA ligase family 2 is selected from one or more of RNA ligases shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4;
the sense strand substrate fragment comprises 2 or 3, and the antisense strand substrate fragment comprises 2 or 3;
The length of the sense strand substrate fragment is 5-12nt;
the antisense strand substrate fragment has a length of 3-14nt.
2. The method of making according to claim 1, 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.
3. The method according to claim 2, wherein 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;
The nucleotide sequence of the first antisense strand substrate fragment is shown as SEQ ID NO. 11, and the nucleotide sequence of the second antisense strand substrate fragment is shown as SEQ ID NO. 12;
SEQ ID NO:9:GAmGAmAUmAUmUUmCAmC;
SEQ ID NO:10:CmCUmUCmA;
SEQ ID NO:11:AUmAUmUCmUCm;
SEQ ID NO:12:UmGAmAGmGGmUGmAAm
Ribonucleotides at positions 2, 4, 6, 8, 10 and 12 of the first sense strand substrate fragment have 2' methoxy modification, a hydroxyl group is at the 5' end, and a hydroxyl group is at the 3' end;
The 1 st, 3 rd and 5 th ribonucleotides of the second sense strand substrate fragment have 2' methoxy modification, the 5' end is a phosphate group, and the 3' end is a hydroxyl group;
The ribonucleotides at positions 2, 4, 6 and 8 of the first antisense strand substrate fragment have 2' methoxy modification, a phosphate group is arranged at the 5' end, and a hydroxyl group is arranged at the 3' end;
the ribonucleotides at positions 1, 3, 5,7, 9 and 11 of the second antisense strand substrate fragment have 2' methoxy modification, the 5' end is a hydroxyl group, and the 3' end is a hydroxyl group.
4. The method of claim 2, wherein in S1), the nucleotide sequence of the first sense strand substrate fragment is set forth in nucleotide sequence SEQ ID No. 13, and the nucleotide sequence of the second sense strand substrate fragment is set forth in nucleotide sequence SEQ ID No. 14;
The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 15, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 16;
SEQ ID NO:13:GAmGAmAUmAUm;
SEQ ID NO:14:UUmCAmCCmCUmUCmA;
SEQ ID NO:15:UmUCmUCm;
SEQ ID NO:16:UmGAmAGmGGmUGmAAmAUmA;
the ribonucleotides at positions 2, 4, 6 and 8 of the first sense strand substrate fragment have 2' methoxy modification, a hydroxyl group is at the 5' end, and a hydroxyl group is at the 3' end;
The ribonucleotides at positions 2, 4, 6, 8 and 10 of the second sense strand substrate fragment have 2' methoxy modification, a phosphate group is arranged at the 5' end, and a hydroxyl group is arranged at the 3' end;
The 1 st, 3 rd and 5 th ribonucleotides of the first antisense strand substrate fragment have 2' methoxy modification, the 5' end is a phosphate group, and the 3' end is a hydroxyl group;
the ribonucleotides at positions 1, 3, 5, 7, 9, 11 and 13 of the second antisense strand substrate fragment have 2' methoxy modification, the 5' end is a hydroxyl group, and the 3' end is a hydroxyl group.
5. The method according to claim 2, 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, and the 3 '-end of the first antisense strand substrate fragment and the 5' -end of the second antisense strand substrate fragment are linked under the catalysis of the RNA ligase to form the antisense strand.
6. The method of claim 5, wherein the first sense strand substrate fragment has a hydroxyl group at the 5 'end and a hydroxyl group at the 3' end, and the second sense strand substrate fragment has a phosphate group at the 5 'end and a hydroxyl group at the 3' end;
The 5 'end of the first antisense strand substrate fragment is a hydroxyl group, 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 of the second antisense strand substrate fragment is a hydroxyl group.
7. The method of claim 1, 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.
8. The method of claim 7, wherein the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3, 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 sense strand substrate fragment, and the RNA ligase;
S2) under the catalysis of the RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment and the third sense strand substrate fragment are connected to form the sense strand, the first antisense strand substrate fragment, the second antisense strand substrate fragment and the third antisense strand substrate fragment are connected to form the antisense strand, and the sense strand and the antisense strand are formed into the QPI-1002 through base complementary pairing.
9. The method according to claim 8, wherein in S1), the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 17;
The nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 18;
The nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 19;
the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 20;
the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 21;
the nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO. 22;
SEQ ID NO:17:GAmGAmAUm;
SEQ ID NO:18:AUmUUmCAmC;
SEQ ID NO:19:CmCUmUCmA;
SEQ ID NO:20:CmUCm;
SEQ ID NO:21:AAmAUmAUmU;
SEQ ID NO:22:UmGAmAGmGGmUGm;
The ribonucleotides at positions 2, 4 and 6 of the first sense strand substrate fragment have 2' methoxy modification, the 5' end is a hydroxyl group, and the 3' end is a hydroxyl group;
The ribonucleotides at positions 2, 4 and 6 of the second sense strand substrate fragment have 2' methoxy modification, a phosphate group is arranged at the 5' end, and a hydroxyl group is arranged at the 3' end;
the 1 st, 3 rd and 5 th ribonucleotides of the third sense strand substrate fragment have 2' methoxy modification, the 5' end is a phosphate group, and the 3' end is a hydroxyl group;
The 1 st and 3 rd ribonucleotides of the first antisense strand substrate fragment have 2' methoxy modification, a phosphate group is arranged at the 5' end, and a hydroxyl group is arranged at the 3' end;
the ribonucleotides at positions 2, 4 and 6 of the second antisense strand substrate fragment have 2' methoxy modification, a phosphate group is arranged at the 5' end, and a hydroxyl group is arranged at the 3' end;
The ribonucleotides at positions 1, 3, 5, 7 and 9 of the third antisense strand substrate fragment have 2' methoxy modification, a hydroxyl group at the 5' end and a hydroxyl group at the 3' end.
10. The method of any one of claims 1, 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.
11. The preparation method according to claim 1, wherein the reaction temperature of the preparation method is 4 ℃ to 37 ℃;
The reaction time of the preparation method is 0.5-24 hours;
The pH of the preparation method is 6-8.5.
CN202410921728.1A 2024-07-10 2024-07-10 A preparation method of QPI-1002 Active CN118460648B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410921728.1A CN118460648B (en) 2024-07-10 2024-07-10 A preparation method of QPI-1002

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202410921728.1A CN118460648B (en) 2024-07-10 2024-07-10 A preparation method of QPI-1002

Publications (2)

Publication Number Publication Date
CN118460648A CN118460648A (en) 2024-08-09
CN118460648B true CN118460648B (en) 2025-01-28

Family

ID=92162322

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410921728.1A Active CN118460648B (en) 2024-07-10 2024-07-10 A preparation method of QPI-1002

Country Status (1)

Country Link
CN (1) CN118460648B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112410336A (en) * 2014-05-29 2021-02-26 夸克制药公司 Methods and compositions for preventing ischemia-reperfusion injury in an organ
CN118166051A (en) * 2022-12-08 2024-06-11 凯莱英生命科学技术(天津)有限公司 Method for synthesizing double-stranded RNA and application of RNA ligase in double-stranded RNA synthesis

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021157730A1 (en) * 2020-02-06 2021-08-12 株式会社Ｖｅｒｉｔａｓ Ｉｎ Ｓｉｌｉｃｏ Nucleic acid drug and use thereof
CN117660374A (en) * 2022-09-08 2024-03-08 凯莱英医药集团（天津）股份有限公司 Application of RNA ligase in oligonucleotide preparation
CN117070583B (en) * 2023-10-16 2024-05-14 吉林凯莱英制药有限公司 Preparation method of siRNA for inhibiting PCSK9 gene expression
CN117757787A (en) * 2023-11-30 2024-03-26 上海兆维科技发展有限公司 Composition for synthesizing nucleotide chain and method for synthesizing nucleotide chain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112410336A (en) * 2014-05-29 2021-02-26 夸克制药公司 Methods and compositions for preventing ischemia-reperfusion injury in an organ
CN118166051A (en) * 2022-12-08 2024-06-11 凯莱英生命科学技术(天津)有限公司 Method for synthesizing double-stranded RNA and application of RNA ligase in double-stranded RNA synthesis

Also Published As

Publication number Publication date
CN118460648A (en) 2024-08-09

Similar Documents

Publication Publication Date Title
Erdmann et al. The involvement of 5S RNA in the binding of tRNA to ribosomes
Ferris et al. Oligomerization of ribonucleotides on montmorillonite: reaction of the 5′-phosphorimidazolide of adenosine
CN110637086B (en) Methods for producing complexes of RNA molecules and peptides and their utilization
CN117070583B (en) Preparation method of siRNA for inhibiting PCSK9 gene expression
WO2024119625A1 (en) Synthesis method for double-stranded rna and use of rna ligase in double-stranded rna synthesis
JP6057185B2 (en) Nucleic acid linker
Heinemann et al. Crystallographic Study of Mechanism of Ribonuclease Ti-Catalysed Specific RNA Hydrolysis
CN117070512A (en) tRNAs and methods of biosynthesis thereof
CN110678549B (en) A method to activate p21 gene expression
CN117070514B (en) Preparation method of non-natural RNA and product
CN117070513B (en) RNA containing phosphorothioate bond and preparation method thereof
CN118460648B (en) A preparation method of QPI-1002
CN118460647B (en) Preparation method of PATISIRAN
Uhlenbeck et al. Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes
CN118460654B (en) A preparation method of Givosiran
EP2256188B1 (en) Method for increasing enzymatic reactivity
US20220056425A1 (en) Rna polymerase for synthesis of modified rna
CN118460651B (en) Method for preparing CEMDISIRAN
CN118460649B (en) A kind of preparation method of Vutrisiran
CN118460652B (en) Preparation method of siRNA for treating non-arterial ischemic optic neuropathy
CN118460653B (en) Preparation method of siRNA for treating alpha 1-antitrypsin deficiency
CN118460655B (en) Preparation method of siRNA for inhibiting FXI gene expression
CN118460656B (en) A preparation method of Lumasiran
CN118460650B (en) A preparation method of Nedosiran
AU2005329833A1 (en) High-affinity RNA aptamer molecule for glutathione S-transferase protein

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant