WO2023138082A1 - 一种真核生物来源的Argonaute蛋白及其应用 - Google Patents

一种真核生物来源的Argonaute蛋白及其应用 Download PDF

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WO2023138082A1
WO2023138082A1 PCT/CN2022/119596 CN2022119596W WO2023138082A1 WO 2023138082 A1 WO2023138082 A1 WO 2023138082A1 CN 2022119596 W CN2022119596 W CN 2022119596W WO 2023138082 A1 WO2023138082 A1 WO 2023138082A1
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guide
rna
eago
protein
dna
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French (fr)
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马立新
何如怡
孙宝彤
王飞
王亚平
李忠臣
颜光波
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湖北大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention belongs to the technical field of molecular biology, and in particular relates to an Argonaute protein derived from eukaryotic organisms and an application thereof.
  • RNA interference RNA interference
  • RISC RNA-induced silencing complex
  • eukaryotic Argonaute protein can cleave RNA with single-stranded DNA as a guide, its DNA cleaving activity has not been found.
  • the purpose of the present invention is to provide an Argonaute protein derived from eukaryotic organisms, and it is found that the Argonaute protein has DNA cutting activity, which is expected to be applied to mammalian cell gene editing.
  • the present invention provides a kind of Argonaute protein (hereinafter referred to as eAgo protein) of eukaryotic origin, and described protein is following any:
  • amino acid sequence is as shown in SEQ ID NO.1; A2).
  • the protein whose amino acid sequence is shown in SEQ ID NO.1 is derived from Thermothelomyces thermophilus, and is named TteAgo protein.
  • the eAgo protein can be artificially synthesized or extracted from natural proteins.
  • the eAgo protein has nuclease activity at 10-60°C; further, the eAgo protein has nuclease activity at 25-55°C; further, the eAgo protein has nuclease activity at 37°C.
  • the eAgo protein may also lose its nuclease activity through mutation.
  • the present invention provides nucleic acid molecules encoding said eAgo protein.
  • nucleic acid molecule is any of the following:
  • the present invention provides an eAgo complex, which is formed by complexing the eAgo protein with a guide molecule, and the guide molecule is an ssDNA guide or an RNA guide.
  • the guide molecule is a 5' end phosphorylated RNA guide, or a 5' end hydroxylated RNA guide, or a 5' end phosphorylated ssDNA guide, or a 5' end hydroxylated ssDNA guide.
  • the length of the ssDNA guide is 12 to 40 nucleotides; more preferably, the length of the ssDNA guide is 12 to 30 nucleotides; optimally, the length of the ssDNA guide is 15 to 20 nucleotides, such as 16, 17 or 18 nucleotides.
  • the present invention when the present invention provides that the eAgo or eAgo complex has nuclease activity, it can specifically cut target nucleic acid in vivo or in vitro, and the target nucleic acid is target RNA or target DNA.
  • the target RNA has no high-level structure, or has a high-level structure, or is double-stranded RNA, or is RNA transcribed in vitro, or is viral genome RNA, or is messenger RNA, namely mRNA, or is other RNA in the cell.
  • the target DNA is synthetic single-strand DNA or double-strand DNA; it can be cell genome DNA or other DNA in the cell.
  • the eAgo or eAgo complex has nuclease activity in a divalent metal cation solution, and the cation is at least one cation selected from Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Mg 2+ , Mn 2+ , and Ca 2+ .
  • the cation is Mn 2+ and/or Mg 2+ .
  • the cation is Mn 2+ .
  • the nuclease activity of the eAgo or eAgo complex has single-base or/and double-base specificity.
  • eAgo or eAgo complex in vivo or in vitro specific cutting of target RNA or target DNA can be divided into the following four types:
  • the eAgo or eAgo complex cuts the target RNA in vitro, and its application process can be: eAgo is mixed with a guide molecule to form an eAgo complex, and the guide molecule is ssDNA or RNA; the eAgo complex is contacted with the target RNA, and the target RNA contains a nucleotide sequence that is mostly complementary to the guide molecule sequence, and the eAgo-guide complex cuts the target RNA at a specific site.
  • the eAgo or eAgo complex cuts the target DNA in vitro, and its application process can be: eAgo is mixed with the RNA guide to form an eAgo complex; the eAgo complex is contacted with the target DNA, the target DNA contains a nucleotide sequence that is mostly complementary to the RNA guide sequence, and the eAgo-guide complex cuts the target DNA at a specific site.
  • the eAgo or eAgo complex cuts the target RNA in the cell, and its application process can be: mix eAgo with a guide molecule to form an eAgo complex, and the guide molecule is ssDNA guide or RNA guide; the eAgo complex is transferred into the cell by transformation, transfection or transduction, and an RNA (i.e., target RNA) in the cell contains a nucleotide sequence that is mostly complementary to the guide molecule sequence.
  • the eAgo or eAgo complex cuts the target DNA in the cell, and its application process can be: eAgo is mixed with the RNA guide to form an eAgo complex; the eAgo complex is transferred into the cell by transformation, transfection or transduction, and a DNA in the cell (i.e., the target DNA) contains a nucleotide sequence that is mostly complementary to the RNA guide sequence.
  • the target RNA or target DNA contains a nucleotide sequence complementary to the RNA guide or ssDNA guide sequence, which means that the guide molecule is either completely complementary to the sequence of the same length contained in the target RNA or target DNA, or there are many mismatches (usually separated, or continuous), and the number of mismatches may be 1, 2, 3, 4 or 5, etc.
  • the target RNA or target DNA contains a nucleotide sequence that is at least 12 bases complementary to the RNA guide or ssDNA guide sequence.
  • the cell is in situ when the target RNA or target DNA is cleaved within the cell.
  • the present invention provides an expression vector containing the nucleic acid molecule provided in the second aspect.
  • the sixth aspect of the present invention provides the application of the expression vector in site-specific modification of genetic material of cells.
  • the method of the application is: introducing the expression vector into the cell, and simultaneously or differently introducing one or more RNA guides, or introducing one or more DNA guides; expressing one or more eAgo proteins in the cell.
  • multiple eAgo proteins are encoded by one expression vector.
  • the expression vector is contained in a viral vector; more preferably, the viral vector is a lentiviral vector or a retroviral vector.
  • the cells are isolated cells.
  • the cells are in situ cells, specifically living tissues, organs or animal cells including human.
  • the cells are eukaryotic cells.
  • the present invention provides a kit comprising eAgo protein, at least one ssDNA guide and/or RNA guide.
  • the present invention provides another kit, which includes the expression vector, at least one ssDNA guide and/or RNA guide.
  • ssDNA guide or RNA guide in the kit refers to the eAgo complex.
  • the present invention provides Argonaute (eAgo) polypeptide derived from eukaryotes that can cut target nucleotide sequences under the guidance of nucleic acid chains, and proves that TteAgo from the fungus Myceliophthora thermophila not only has the activity of cutting RNA but also has the activity of cutting DNA, and proposes the application potential of eAgos in DNA targeted editing.
  • eAgo Argonaute
  • the present invention provides an expression vector comprising the nucleic acid encoding the polypeptide, as well as a composition, a kit and an application method for cutting and editing a target nucleic acid in a sequence-specific manner.
  • the polypeptide, nucleic acid, expression vector, composition, kit and method of the present invention can perform site-specific modification of intracellular and extracellular genetic material, so it can be effectively applied to many fields of biotechnology, such as nucleic acid detection, gene editing and gene modification, etc., and provides a new tool for gene editing, modification and molecular detection of Argonaute polypeptides based on eukaryotic origin.
  • the protein provided by the invention has binding activity to RNA guides and single-stranded DNA (ssDNA) guides, and has nuclease activity to target RNA and target DNA, so that when RNA guides or ssDNA guides with most of the paired sequences of target RNA or target DNA combine with eAgo to form eAgo-guide complexes, and when the eAgo-guide complexes associate with target RNA or target DNA, site-specific cleavage of target RNA or target DNA occurs. And site specificity can be adjusted by selecting RNA or ssDNA guides with specific nucleotide sequences.
  • the eAgo used in the present invention can use 16-18nt RNA and/or DNA guide to specifically cut target RNA and/or target DNA, especially when ssDNA is used as a guide to cut RNA, it also has high activity, and DNA guide has a shorter cycle and lower price than RNA synthesis, which can greatly save costs.
  • the eAgo used in the present invention does not rely on special motifs near the target site to recognize and bind to the target, and the design of the DNA guide is convenient without considering site restrictions.
  • the eAgo used in the present invention has strong cleavage activity, strictly depends on the complementary pairing of the guide and the target to exert cleavage activity, does not have non-specific "incidental cleavage" activity of CRISPR-related proteins, and has better specificity. Further, by mutating the active site of eAgo, an eAgo that completely loses its cleavage activity can be obtained, which can be fused with other effector proteins, further expanding its application.
  • Fig. 1 is a schematic diagram of the phylogenetic tree of some characterized Ago proteins provided by the present invention
  • Figure 2 is a schematic diagram of the sequence alignment of fourteen characterized Ago proteins provided by the present invention.
  • Fig. 3 is the SDS-PAGE gel figure of TteAgo protein among the embodiment 1, and wherein, swimming lane 1 is total protein, swimming lane 2 is broken bacteria supernatant, swimming lane 3 is 200mM imidazole eluent, swimming lane 4 and swimming lane 5 are agarose beads after Im7 incubation, swimming lane 6 and swimming lane 7 are the supernatant after 3C protease digestion;
  • Fig. 4 is the schematic diagram of RNA guide, DNA guide, target RNA, target DNA used for testing in embodiment 2, and the urea/polyacrylamide gel electrophoresis figure of the product of TteAgo cutting target RNA and target DNA;
  • Fig. 5 is the urea/polyacrylamide gel electrophoresis graph of the products of different length guide-mediated TteAgo cutting target RNA or target DNA in Example 3, wherein, A to C are successively the gel electrophoresis graphs of different lengths of RNA guide cutting target RNA, different lengths of DNA guide cutting target RNA, and different lengths of RNA guide cutting target DNA;
  • Fig. 6 is the urea/polyacrylamide gel electrophoresis figure of guide-mediated TteAgo cutting target RNA or target DNA product under different metal ion conditions in Example 4;
  • Fig. 7 is the urea/polyacrylamide gel electrophoresis figure of guide-mediated TteAgo cutting target RNA and target DNA product under different Mn 2+ or Mg 2+ ion concentration conditions in Example 4;
  • Fig. 8 is the urea/polyacrylamide gel electrophoresis figure of RNA guide mediation TteAgo cutting target RNA and target DNA product under different temperature conditions in embodiment 5;
  • FIG. 9 is a schematic diagram of RNA guides for single-base and double-base mutations and DNA guides for single-base mutations in Example 6;
  • Fig. 10 is the urea/polyacrylamide gel electrophoresis graph of TteAgo cleavage target RNA or target DNA mediated by RNA guides with single base and double base mutations and DNA guides with single base mutations in Example 6.
  • the present invention provides a kind of eAgo protein of eukaryotic origin, and described eAgo protein is any of the following:
  • TteAgo protein The amino acid sequence of the protein shown in SEQ ID NO.1, the protein derived from Myceliophthora thermophilic eukaryotic microorganism Thermothelomyces thermophilus, named TteAgo protein, the sequence of the nucleic acid molecule encoding the TteAgo protein is shown in SEQ ID NO.1.
  • the sequence of nucleic acid analysis encoding this type of protein is: a polynucleotide sequence that hybridizes to the DNA molecule shown in SEQ ID NO.3 under stringent conditions, or a nucleotide sequence that has at least 50%, at least 80%, at least 90% or at least 95% sequence identity with the sequence shown in SEQ ID NO.3.
  • the eAgo protein has binding activity to RNA guides and ssDNA guides, and has nuclease activity to target RNA and target DNA, so that when the RNA guide or ssDNA guide having most of the pairing with the target RNA or DNA sequence is combined with the eAgo protein to form an eAgo complex, and when the eAgo-guide complex associates with the target RNA or DNA, the target DNA or RNA can undergo site-specific cleavage.
  • the guide molecule can specifically be phosphorylated RNA and/or ssDNA at the 5' end, or hydroxylated RNA and/or ssDNA; the guide molecule can include a terminal 5'-triphosphate.
  • the ssDNA guide is 12 to 30 nucleotides in length, even preferably 15 to 20 nucleotides, such as 16, 17 or 18 nucleotides in length.
  • the eAgo protein has nuclease activity at a temperature range of 25-65°C; advantageously and preferably, the eAgo protein of the present invention has nuclease activity at 37°C.
  • the nuclease activity of the eAgo protein requires the presence of cations, and the cations are any one or any combination of Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Mg 2+ , Mn 2+ , Ca 2+ ; particularly preferably, the cations are Mn 2+ and Mg 2+ .
  • the concentration of the cation may vary from about 0.01 mM to about 2000 mM; particularly preferably, the range is from about 0.05 mM to about 20 mM.
  • the 5' end and/or 3' end of the eAgo protein has multiple nuclear localization sequences (nuclear localization sequence, NLS).
  • the target RNA is free of higher order structure. In other embodiments, the target RNA is of higher order structure.
  • Other possible target RNAs include double-stranded RNA, in vitro transcribed RNA, viral genomic RNA, messenger RNA (mRNA) and other RNAs within cells.
  • the length of the eAgo protein in the present invention can be 1082 amino acids shown in SEQ ID NO.1, or it can be a longer or shorter continuous amino acid.
  • the number of amino acids (longer or shorter) may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 .
  • the definition of the above-mentioned continuous amino acids includes or is less than the full length of the eAgo protein of the present invention (1082 amino acids), but retains a functional fragment that forms an eAgo-guide complex with the guide molecule and has site-specific cleavage activity on target RNA and/or target DNA.
  • the eAgo protein with nuclease activity and the eAgo complex can specifically cut target RNA or target DNA in vivo or in an in vitro environment, and the internal body is in a cell.
  • the present invention also provides a method for site-specifically modifying the genetic material of cells, specifically: introducing an expression vector containing a polynucleotide sequence encoding the eAgo protein into the cell, and simultaneously or at different times introducing one or more RNA and/or ssDNA guides, thereby expressing the eAgo protein in the cell.
  • the site-specific modification methods occur in isolated cells; in other embodiments, the methods of the invention may occur in cells in situ, which may be living tissues, organs or animals including humans.
  • the eAgo protein of the site-specific modification method may be encoded by one expression vector, and in other such methods, one or more eAgo proteins may be encoded by two expression vectors; in some embodiments, the one expression vector may encode all eAgos proteins.
  • the expression vector described in the site-specific modification method may be contained in a viral vector, such as a lentiviral vector or a retroviral vector.
  • the site-specific modification method may be used in eukaryotic cells.
  • the kit provided by the invention comprises the following three kinds:
  • Kit one includes: the eAgo protein of the present invention, and RNA guide and/or ssDNA guide;
  • the second kit includes: an expression vector containing the polynucleotide sequence encoding the eAgo protein, RNA guide and/or ssDNA guide;
  • Kit three includes: a viral vector containing the expression vector, a viral vector encoding RNA guide and/or ssDNA guide.
  • the eAgo protein of the present invention has binding activity to the RNA guide or ssDNA guide, but has no nuclease activity on the target DNA and target RNA
  • the RNA guide or ssDNA guide that has most of the pairing with the target RNA or target DNA combines with the eAgo protein to form an eAgo complex
  • the eAgo-guide complex associates with the target RNA or target DNA, the target RNA or target DNA is site-specifically hindered.
  • the preparation method of the eAgo protein without nuclease activity can be: by mutating one or more amino acid residues essential to the catalytic activity of the eAgo protein to cause new nuclease activity, especially the loss of endonuclease activity. That is, at least one amino acid located in an evolutionarily conserved amino acid quadruple (DEDD) is mutated.
  • a mutation may be a single amino acid change in any one or more of the following amino acid sequence portions of the TteAgo protein:
  • an amino acid change is a single change at one or more of the above highlighted residues.
  • the single mutation is a non-conservative substitution, such as from D to A, or from E to A. Therefore, any substitution other than D to E or E to D is possible.
  • nuclease-deficient eAgo in the present invention may also include any structural changes as defined above for eAgos having nuclease activity. For example, the extent of sequence identity compared to the reference sequence, the composition of the eAgo in terms of amino acid domains, and the overall length in terms of amino acids.
  • the definition of guides is similar to that used by eAgos with nuclease activity in the present invention.
  • eAgo complexes that do not have nuclease activity
  • site-specific blockade provides a precise means to block transcription of a gene of interest, or to block, disrupt or interfere with specific sites involved in the regulation of gene expression.
  • the present invention provides a method for site-specific targeted blocking of target nucleic acids in cells, comprising the following steps: mixing nuclease-free eAgo with RNA guides or ssDNA guides to form eAgo complexes; transferring the eAgo complexes into cells (such as by transformation, transfection, fiber injection, etc.), and the guide sequence is basically complementary to a nucleotide sequence contained in the target nucleic acid.
  • the method for site-specific targeted blocking of target nucleic acid in cells can also adopt the following steps: transfect, transform or transduce cells with an expression vector containing the eAgo encoding the nuclease-free activity; transfect, transform or transduce the first RNA guide or ssDNA guide sequence and the second ssRNA guide or ssDNA guide sequence; wherein the sequence of at least one guide molecule is basically complementary to the nucleotide sequence contained in the target nucleic acid, and the eAgo produced by expressing in the cell and the guide molecule form an eAgo complex capable of blocking a specific site .
  • the method of site-specific blocking of target polynucleotides using said nuclease-free eAgos can target disruption of gene expression and/or control elements of gene expression, such as promoters or enhancers.
  • nuclease activity eAgos defined in the present invention.
  • Embodiment 1 TteAgo expression and purification
  • the purified protein was collected and identified by SDS-polyacrylamide gel for purity, and ultrafiltered to Buffer B (20mM Tris–HCl pH 7.4, 500mM NaCl, 1mM TCEP). The protein was divided into small portions, frozen in liquid nitrogen, and stored at -80°C.
  • FIG. 2 shows the region where the catalytic DEDX quadruplex is located, and the sequence identity between TteAgo and other Agos.
  • Figure 3 shows the results of gel analysis of TteAgo after purification of TteAgo by Ni-NTA column and molecular sieves. Calculated by http://www.expasy.org/, the expected size of TteAgo is about 118 kDa.
  • Lysis assays were all performed at 37°C in a 5:2:1 (TteAgo:guide:target) molar ratio.
  • 1 uM TteAgo was mixed with 400 nM guide in a reaction buffer containing 10 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 5 mM MnCl 2 and 5% glycerol, and incubated at 37°C for 10 minutes for guide loading.
  • Nucleic acid targets were added to a final concentration of 200 nM.
  • Figures A and B in Figure 4 are schematic diagrams of RNA guides, DNA guides, target RNAs, and target DNAs used for testing, and arrows indicate predicted cleavage sites.
  • Figures C and D in Figure 4 are urea/polyacrylamide gel electrophoresis images of the products of TteAgo cutting target RNA and target DNA.
  • TteAgo catalytic quadruplex DEDD were mutated to amino acid A, and the double mutant DM was recorded as TteAgo-DM. From Figure 4C and 4D, it can be seen that TteAgo-DM lost the activity of DNA guide cutting target RNA and target DNA.
  • Embodiment 3 The influence of guide molecule length on cutting effect
  • RNA guides or DNA guides of different lengths were combined with TteAgo to verify its activity of cutting target RNA or target DNA.
  • Figure A in Figure 5 shows that under the guide condition of 5' phosphorylated RNA with a length of 12-30nt, TteAgo showed guide-guided target RNA cleavage within 30 minutes;
  • Figure B in Figure 5 showed that under the condition of a 5' phosphorylated DNA guide with a length of 12-30nt, TteAgo showed guide-guided target RNA cleavage within 30 minutes;
  • Ago exhibits guide-directed cleavage of target ssDNA.
  • the target RNA can be effectively cut when the RNA guide length ranges from 12-25nt and the DNA guide length ranges from 12-30nt; the target ssDNA can be effectively cut when the RNA guide length ranges from 12-30nt.
  • test results are shown in Figure 6. It can be seen that when the cation is Mn 2+ , Mg 2+ , Co 2+ or/and Ni 2+ , TteAgo combined with RNA guide or DNA guide can effectively cut target RNA ( Figure 6A, 6B, 6C, 6D); when the cation is Mn 2+ , TteAgo combined with RNA guide can effectively cut target DNA ( Figure 6E, 6F).
  • RNA guides with single or double base mutations (m1, m2, m3, m4, m5, m6, m7,
  • RNA guides with single base mutations m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, m12, m13, m14, m15, m16, m17, m18, the specific mutations are shown in Figure 9).
  • the cleavage activity of TteAgo to the target DNA was significantly weakened.
  • the eukaryotic-derived Argonaute protein provided by the present invention has binding activity to RNA guides and ssDNA guides, and has nuclease activity on both target RNA and target DNA, and the eAgo protein of the present invention can perform site-specific modification of intracellular and extracellular genetic material. Therefore, it can be effectively applied to many fields of biotechnology, such as nucleic acid detection, gene editing and gene modification.

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Abstract

提供了一种真核生物来源的Argonaute蛋白及其应用，所述Argonaute蛋白的氨基酸序列如SEQ ID NO.1所示，或与如SEQ ID NO.1所示序列有至少50％的序列一致性；首次证明了真核Argonaute蛋白对DNA的特异性切割活性，为真核Argonaute与DNA的相互作用研究提供了实验证明；同时，使用的多肽、核酸、表达载体、组合物、试剂盒和方法能够对细胞内和细胞外的遗传物质进行位点特异性操作，可有效地应用于生物技术的许多领域，为基于真核生物来源的Argonaute多肽的基因编辑、修饰及分子检测提供了一种新的工具。

Description

一种真核生物来源的Argonaute蛋白及其应用 技术领域
本发明属于分子生物技术领域，具体涉及一种真核生物来源的Argonaute蛋白及其应用。
背景技术
真核Argonaute(简称eAgo)在真核生物的RNA干扰(RNA interference,RNAi)途径中起着关键作用，是RNA诱导沉默复合物(RNA-induced silencing complex,RISC)的主要功能核心，可以与作为互补的RNA靶标特异性识别向导小的单链RNA分子结合，或者通过一些eAgos酶固有的核酸酶活性直接切割靶标，或者招募其他沉默蛋白来作用于靶标，使转录受到抑制。因此，eAgos可以在转录水平调控基因表达，保护宿主免受RNA病毒的入侵，并通过减少转座子的移动性来保持基因组的完整性。最近也有研究表明，一些AGO蛋白除了在典型的RNAi通路中的作用外，还可以通过其它机制调节基因表达，戚益军研究组之前研究发现AGO1也存在于细胞核中，这意味着AGO1可能在细胞核内也有重要功能，但并未解释其具体机制。目前，大多数文献只报道了eAgo在体外特异性的RNA切割的活性，如hAgo2和KpAgo，并未有文献报道eAgo对DNA的特异性切割活性。
长期以来，人们广泛关注eAgo在RNAi途径中的重要作用，并在高等动植物和酵母细胞中均发现了具有高RNase活性的eAgo，但是对真核Argonaute和DNA的相互作用并没有详细的探究，虽然有研究发现真核来源的Argonaute蛋白可以以单链DNA作为向导靶向切割RNA，但并未发现其对DNA的切割活性。同时，关于Argonaute蛋白在基因编辑上的应用，由于来自高等动植物的真核 Argonaute蛋白可能会参与受体细胞内的RNAi途径无法实现有效基因编辑，因此目前并未有利用真核Argonaute蛋白进行基因编辑。
发明内容
有鉴于此，本发明目的在于提供一种真核生物来源的Argonaute蛋白，并发现了该Argonaute蛋白具有对DNA的切割活性，有望应用于哺乳动物细胞基因编辑。
一方面，本发明提供了一种真核生物来源的Argonaute蛋白(以下简称eAgo蛋白)，所述蛋白为如下任一：
A1).氨基酸序列如SEQ ID NO.1所示；A2).与SEQ ID NO.1所示的氨基酸序列具有至少50％、至少80％、至少90％或至少95％的序列一致性且具有相同功能的蛋白质。
具体的，所述氨基酸序列如SEQ ID NO.1所示的蛋白来源于嗜热毁丝霉真核微生物(Thermothelomyces thermophilus)，将其命名为TteAgo蛋白。
优选地，所述eAgo蛋白可以是人工合成的，也可以是提取的天然蛋白。
优选地，所述eAgo蛋白在10～60℃具有核酸酶活性；进一步地，所述eAgo蛋白在25～55℃具有核酸酶活性；更进一步地，所述eAgo蛋白在37℃具有核酸酶活性。
优选地，所述eAgo蛋白也可能通过突变失去核酸酶活性。
第二方面，本发明提供了编码所述eAgo蛋白的核酸分子。
具体地，所述核酸分子为如下任一：
B1).核苷酸序列如SEQ ID NO.3所示的DNA分子；B2).在严格条件下与SEQ ID NO.3所示的DNA分子杂交且编码所述eAgo蛋白的DNA分子；B3).与B1)或B2)限定的DNA序列具有至少50％、至少80％、至少90％或至少95％的序列一致性且编码所述eAgo蛋白的DNA分子。
第三方面，本发明提供了一种eAgo复合物，由所述eAgo蛋白与向导分子复合形成，所述向导分子为ssDNA向导或RNA向导。
优选地，所述向导分子为5’末端磷酸化的RNA向导，或5’末端羟基化的RNA向导，或5’末端磷酸化的ssDNA向导，或5’末端羟基化的ssDNA向导。
优选地，所述ssDNA向导的长度为12至40个核苷酸；更加优选地，所述ssDNA向导的长度为12至30个核苷酸；最优地，所述ssDNA向导的长度为15至20个核苷酸，如16、17或18个核苷酸。
第四方面，当本发明提供了所述eAgo或eAgo复合物具有核酸酶活性时，其能够在体内或体外环境中特异性切割靶核酸，所述靶核酸为靶RNA或靶DNA。
需要说明的是，所述靶RNA没有高级结构，或有高级结构，或为双链RNA，或为体外转录的RNA，或为病毒基因组RNA，或为信使RNA即mRNA，或为细胞内的其他RNA。所述靶DNA为合成的单链DNA，或为双链DNA；可以是细胞基因组DNA，还可以是细胞内的其他DNA。
优选地，所述eAgo或eAgo复合物在二价金属阳离子溶液中具有核酸酶活性，所述阳离子为Fe 2+、Co 2+、Ni 2+、Cu 2+、Zn 2+、Mg 2+、Mn 2+、Ca 2+中的至少一种阳离子。
更为优选地，所述阳离子为Mn 2+和/或Mg 2+。
最优地，所述阳离子为Mn 2+。
优选地，所述eAgo或eAgo复合物的核酸酶活性具有单碱基或/和双碱基专一性。
具体的，所述eAgo或eAgo复合物在体内或体外特异性切割靶RNA或靶DNA中的应用可分为如下四种类型：
(1)所述eAgo或eAgo复合物在体外切割靶RNA，其应用过程可为：将eAgo与向导分子混合，形成eAgo复合物，所述向导分子为ssDNA或RNA；令eAgo复合物与靶RNA接触，所述靶RNA含有与所述向导分子序列大部分互补 的核苷酸序列，所述eAgo-向导复合物在特定位点切割靶RNA。
(2)所述eAgo或eAgo复合物在体外切割靶DNA，其应用过程可为：将eAgo与RNA向导混合，形成eAgo复合物；令eAgo复合物与靶DNA接触，所述靶DNA含有与所述RNA向导序列大部分互补的核苷酸序列，所述eAgo-向导复合物在特定位点切割靶DNA。
(3)所述eAgo或eAgo复合物在细胞内切割靶RNA，其应用过程可为：将eAgo与向导分子混合，形成eAgo复合物，所述向导分子为ssDNA向导或RNA向导；通过转化、转染或转导将eAgo复合物转入细胞，细胞内的一个RNA(即靶RNA)含有与所述向导分子序列大部分互补的核苷酸序列。
(4)所述eAgo或eAgo复合物在细胞内切割靶DNA，其应用过程可为：将eAgo与RNA向导混合，形成eAgo复合物；通过转化、转染或转导将eAgo复合物转入细胞，细胞内的一个DNA(即靶DNA)含有与所述RNA向导序列大部分互补的核苷酸序列。
需要说明的是，靶RNA或靶DNA含有与RNA向导或ssDNA向导序列互补的核苷酸序列，这意味着所述向导分子要么与靶RNA或靶DNA包含的相同长度的序列完全互补，要么存在许多错配(通常是分离的，也可能是连续的)，错配的数目可能是1、2、3、4或5等。
优选地，所述靶RNA或靶DNA含有与所述RNA向导或ssDNA向导序列至少12个碱基互补的核苷酸序列。
优选地，在所述细胞内切割靶RNA或靶DNA时，所述细胞为原位细胞。
第五方面，本发明提供了一种表达载体，所述表达载体含有第二方面提供的核酸分子。
进一步地，本发明第六方面提供了所述表达载体在位点特异性修饰细胞的遗传材料中的应用。
优选地，所述应用的方法为：将表达载体导入细胞，并同时或不同时导入一 个或多个RNA向导，或导入一个或多个DNA向导；在细胞内表达一种或多种所述eAgo蛋白。
优选地，多种eAgo蛋白由一个表达载体编码。
优选地，所述表达载体包含在病毒载体中；更加优选地，所述病毒载体为慢病毒载体或逆转录病毒载体。
优选地，所述细胞为分离的细胞。
优选地，所述细胞为原位细胞，具体可为活体组织、器官或包括人在内的动物细胞。
优选地，所述细胞为真核生物细胞。
第七方面，本发明提供了一种试剂盒，所述试剂盒包含eAgo蛋白、至少一个ssDNA向导和/或RNA向导。
第八方面，本发明提供了另一种试剂盒，所述试剂盒包括所述表达载体、至少一个ssDNA向导和/或RNA向导。
需要说明的是，试剂盒中ssDNA向导或RNA向导的选取参照eAgo复合物。
本发明的有益效果为：
本发明提供了能在核酸链的引导下切割靶核苷酸序列的真核生物来源的Argonaute(eAgo)多肽，并证明了来自真菌嗜热毁丝霉的TteAgo不仅具有切割RNA的活性也具有切割DNA的活性，提出了eAgos在DNA靶向编辑上的应用潜力。
本发明提供了包含编码所述多肽的核酸的表达载体，以及用于以序列特异性方式切割和编辑靶核酸的组合物、试剂盒和应用方法。本发明的多肽、核酸、表达载体、组合物、试剂盒和方法能够对细胞内和细胞外的遗传物质进行位点特异性修饰，因此可有效地应用于生物技术的许多领域，比如核酸检测、基因编辑和基因修饰等，为基于真核生物来源的Argonaute多肽的基因编辑、修饰及分子检测提供了一种新的工具。
本发明提供的蛋白对RNA向导和单链DNA(ssDNA)向导有结合活性，并且具有对靶RNA和靶DNA的核酸酶活性，从而当与靶RNA或靶DNA序列具有大部分配对的RNA向导或ssDNA向导与eAgo结合形成eAgo-向导复合物时，并且当eAgo-向导复合物与靶RNA或靶DNA缔合时，靶RNA或靶DNA发生位点特异性切割。且可以通过选择具有特定核苷酸序列的RNA或ssDNA向导来调节位点特异性。
本发明用到的eAgo能够使用长度为16-18nt的RNA和/或DNA向导特异性切割靶RNA和/或靶DNA，特别是在以ssDNA作为向导切割RNA时也具有很高的活性，而DNA向导相对于RNA合成周期短、价格低廉，能极大地节省成本。
此外，本发明用到的eAgo不用依赖靶位点附近的特殊基序来识别和结合靶，DNA向导设计方便，不用考虑位点限制。
本发明用到的eAgo切割活性强，严格依赖于向导和靶的互补配对发挥切割活性，不存在CRISPR相关蛋白的非特异性“附带切割”活性，特异性更好。进一步地，将eAgo的活性位点进行突变，能够得到完全丧失切割活性的eAgo，可以融合其它效应蛋白，进一步拓展了其应用。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图做简单的介绍，显而易见地，下面所描述的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明提供的部分已表征Ago蛋白的进化树示意图；
图2是本发明提供的十四个已表征Ago蛋白的序列比对示意图；
图3是实施例1中TteAgo蛋白的SDS-PAGE凝胶图，其中，泳道1为总蛋白，泳道2为破菌上清，泳道3为200mM咪唑洗脱液，泳道4和泳道5均为Im7 孵育后琼脂糖珠，泳道6和泳道7为3C蛋白酶酶切后的上清；
图4是实施例2中用于测试的RNA向导、DNA向导、靶RNA、靶DNA的示意图，以及TteAgo切割靶RNA和靶DNA的产物的尿素/聚丙烯酰胺凝胶电泳图；
图5是实施例3中不同长度的向导介导TteAgo切割靶RNA或靶DNA的产物的尿素/聚丙烯酰胺凝胶电泳图，其中，A至C依次为不同长度的RNA向导切割靶RNA、不同长度的DNA向导切割靶RNA、不同长度的RNA向导切割靶DNA的凝胶电泳图；
图6是实施例4中不同金属离子条件下向导介导TteAgo切割靶RNA或靶DNA产物的尿素/聚丙烯酰胺凝胶电泳图；
图7是实施例4中不同Mn 2+或Mg 2+离子浓度条件下向导介导TteAgo切割靶RNA和靶DNA产物的尿素/聚丙烯酰胺凝胶电泳图；
图8是实施例5中不同温度条件下RNA向导介导TteAgo切割靶RNA和靶DNA产物的尿素/聚丙烯酰胺凝胶电泳图；
图9是实施例6中单碱基和双碱基突变的RNA向导、单碱基突变的DNA向导的示意图；
图10是实施例6中单碱基和双碱基突变的RNA向导、单碱基突变的DNA向导介导TteAgo切割靶RNA或靶DNA的尿素/聚丙烯酰胺凝胶电泳图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例和附图，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提供了一种真核生物来源的eAgo蛋白，所述eAgo蛋白为以下任一：
i)氨基酸序列如SEQ ID NO.1所示的蛋白质，该蛋白质来源于嗜热毁丝霉真 核微生物Thermothelomyces thermophilus，命名为TteAgo蛋白，编码TteAgo蛋白的核酸分子的序列如SEQ ID NO.1所示。
ii)与SEQ ID NO.1所示的氨基酸序列具有至少50％的序列一致性且具有相同功能的蛋白质；优选地，至少80％；更优地，至少90％；最佳至少95％的序列一致性。编码该类型蛋白的核酸分析的序列为：在严格条件下与SEQ ID NO.3所示的DNA分子杂交的多核苷酸序列，或与SEQ ID NO.3所示序列具有至少50％、至少80％、至少90％或至少95％的序列一致性的核苷酸序列。
优选地，所述eAgo蛋白对RNA向导和ssDNA向导具有结合活性，并且具有对靶RNA和靶DNA的核酸酶活性，从而当与靶RNA或DNA序列具有大部分配对的RNA向导或ssDNA向导与所述eAgo蛋白结合形成eAgo复合物时，并且当eAgo-向导复合物与靶RNA或DNA缔合时，靶DNA或RNA能够发生位点特异性切割。
所述向导分子具体可以是5’末端磷酸化的RNA和/或ssDNA，也可以是羟基化的RNA和/或ssDNA；向导分子可以包含末端5’-三磷酸酯。
优选地，所述ssDNA向导的长度12至30个核苷酸，甚至优选地为15至20个核苷酸，比如16、17或18个核苷酸。
优选地，所述eAgo蛋白在25～65℃的温度范围内具有核酸酶活性；有利地且优选地，本发明的eAgo蛋白在37℃下具有核酸酶活性。
优选地，所述eAgo蛋白的核酸酶活性的发挥需要阳离子的存在，所述阳离子为Fe 2+，Co 2+，Ni 2+，Cu 2+，Zn 2+，Mg 2+，Mn 2+，Ca 2+中的任意一种或任意组合的；特别优选地，所述阳离子为Mn 2+和Mg 2+。所述阳离子的浓度范围可以从约0.01mM至约2000mM变化；特别优选地，范围是约0.05mM至约20mM。
在一些实施例中，所述eAgo蛋白的5’末端和/或3’末端具有多个核定位序列(nuclear localisation sequence，NLS)。
在一些实施方案中，靶RNA是没有高级结构地。在其他实施方案中，靶RNA 是有高级结构地。其他可能的靶RNA包括双链RNA，体外转录的RNA，病毒基因组RNA，信使RNA(mRNA)和细胞内的其他RNA。
具体的说，本发明中所述eAgo蛋白的长度可以是SEQ ID NO.1所示的1082个氨基酸，也可能是一段更长或更短的连续氨基酸。氨基酸个数(更长或更短)可能是1，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20，21，22，23，24，25，26，27，28，29，30，31，32，33，34，35，36，37，38，39，40，41，42，43，44，45，46，47，48，49，50，51，52，53，54，55，56，57，58，59，60，61，62，63，64，65，66，67，68，69，70，71，72，73，74，75，76，77，78，79，80，81，82，83，84，85，86，87，88，89，90，91，92，93，94，95，96，97，98，99，100，101，102，103，104，105，106，107，108，109，110，111，112，113，114，115，116，117，118，119，120，121，122，123，124，125，126，127，128，129，130，131，132，133，134，135，136，137，138，139，140，141，142，143，144，145，146，147，148，149，150，151，152，153，154，155，156，157，158，159，160，161，162，163，164，165，166，167，168，169，170，171，172，173，174，175，176，177，178，179，180，181，182，183，184，185，186，187，188，189，190，191，192，193，194，195，196，197，198，199，200，201，202，203，204，205，206，207，208，209，210，211，212，213，214，215，216，217，218，219，220，221，222，223，224，225，226，227，228，229，230，231，232，233，234，235，236，237，238，239，240，241，242，243，244，245，246，247，248，249，250，251，...(连续数字),和/或1082。
上述连续氨基酸的定义是包括或小于本发明所述eAgo蛋白全长(1082个氨基酸)，但保留了与向导分子形成eAgo-向导复合物和对靶RNA和/或靶DNA的位点特异性切割活性的功能片段。
所述具有核酸酶活性的eAgo蛋白与eAgo复合物可以在体内或体外环境中 特异性切割靶RNA或靶DNA，所述体内为细胞内。
本发明还提供了一种位点特异性修饰细胞的遗传材料的方法，具体为：将一个含有编码所述eAgo蛋白的多核苷酸序列的表达载体导入细胞，并同时或不同时导入一个或多个RNA和/或ssDNA向导，从而在细胞内表达所述eAgo蛋白。
在一些实施案例中，位点特异性修饰方法发生在分离的细胞中；在其他的实施案例中，本发明中的方法可能发生在原位细胞中，可能是活体组织、器官或包括人在内的动物。
所述位点特异性修饰方法的eAgo蛋白可能由一个表达载体编码，在其他此类方法中，一个或多个eAgo蛋白可能由两个表达载体编码；在一些实施案例中，所述一个表达载体可以编码所有eAgos蛋白。
所述位点特异性修饰方法中所述的表达载体可能包含在病毒载体中，如慢病毒载体或逆转录病毒载体。
所述位点特异性修饰方法可能用在真核生物细胞中。
本发明提供的试剂盒包括以下三种：
试剂盒一包含：本发明所述eAgo蛋白，以及RNA向导和/或ssDNA向导；
试剂盒二包含：含有编码所述eAgo蛋白的多核苷酸序列的表达载体，RNA向导和/或ssDNA向导；
试剂盒三包含：包含所述表达载体的病毒载体，一个编码RNA向导和/或ssDNA向导的病毒载体。
当本发明所述eAgo蛋白对RNA向导或ssDNA向导具有结合活性，但对靶DNA和靶RNA没有核酸酶活性时，与靶RNA或靶DNA具有大部分配对的RNA向导或ssDNA向导与eAgo蛋白结合形成eAgo复合物时，并且当所述eAgo-向导复合物与靶RNA或靶DNA缔合时，靶RNA或靶DNA发生位点特异性阻碍。
所述没有核酸酶活性的eAgo蛋白的制备方法可以为：通过突变对eAgo蛋白的催化活性必不可少的一个或多个氨基酸残基来造成新的核酸酶活性，特别是 核酸内切酶活性缺失。也就是说，至少突变一个位于进化保守的氨基酸四联体(DEDD)中的氨基酸。因此，突变可能是TteAgo蛋白中以下任意一个或多个氨基酸序列部分中的单个氨基酸变化：
Figure PCTCN2022119596-appb-000001
更特别地，氨基酸变化是在以上一个或多个突出显示的残基处的单个变化。优选地，单个突变是非保守取代，例如从D到A，或从E到A。因此，除了D到E或E到D以外的任何替换都是可能的。
除了替换以外，一个或多个突出显示的残基可以被简单地删除；可选地，可以连续或不连续地删除氨基酸序列内的一个或多个氨基酸，也可以整体删除一个或多个序列基序。可以进行上述更改的任何组合，例如一个基序非保守变化，其他三个基序缺失。本发明中的核酸酶缺陷型eAgo的结构特征还可以包括如上文关于具有核酸酶活性eAgos所定义的任何结构变化。例如，与参考序列相比的序列一致性范围，就氨基酸结构域而言的eAgo的组成以及就氨基酸而言的总长度。向导的定义与本发明中具有核酸酶活性eAgos所用的向导相似。
对于不具备核酸酶活性的eAgo复合物，这意味着有利地存在一种通过特异性序列识别来阻断靶DNA或RNA中的特定位点的方法，靶可以是单链和双链。这样的位点特异性阻断提供了阻断目标基因转录的精确手段，或者阻断、破坏或干扰参与基因表达调节的特异性位点。
故，本发明提供了一种位点特异性靶向阻断细胞中靶核酸的方法，包括以下步骤：混合无核酸酶活性的eAgo与RNA向导或ssDNA向导，形成eAgo复合物；将eAgo复合物转入细胞(如通过转化、转染、纤维注射等方式)，所述向导序列与靶核酸包含的一段核苷酸序列基本互补。
基于此，在细胞内对靶核酸进行位点特异性靶向阻断的方法还可以采用如下步骤：用含有编码所述无核酸酶活性eAgo的表达载体转染、转化或转导细胞； 转染、转化或转导第一个RNA向导或ssDNA向导序列和第二个ssRNA向导或ssDNA向导序列；其中至少一个向导分子的序列与靶核酸中包含的核苷酸序列基本互补，并且在细胞中表达产生的eAgo和向导分子形成能阻断特定位点eAgo复合物。
优选地，使用所述无核酸酶活性eAgos的位点特异性阻断靶多核苷酸方法可以靶向破坏基因表达和/或基因表达的控制元件，如启动子或增强子。
上述位点特异性阻断靶DNA或靶RNA的各种方法中，特别优选或任选的方面参照本发明定义的核酸酶活性eAgos。
实施例1TteAgo表达和纯化
转化pET28a-CL7-TteAgo质粒至大肠杆菌BL21(DE3)，单菌落接种到含有50μg/mL卡那霉素的LB液体培养基中，于37℃、220rpm的摇床上摇瓶培养，当菌体OD600达到0.8时，移至18℃摇床上，IPTG诱导过夜。6000rpm离心10min收集菌体，用Buffer A(20mM Tris–HCl pH 7.4、500mM NaCl、10mM imidazole)洗菌后，将菌体重悬于Buffer A，添加终浓度1mM的PMSF，高压破碎。18000rpm离心30min，收集上清。上清过滤后，进行Ni-NTA纯化。其中，CL7-TteAgo融合蛋白的氨基酸序列如SEQ ID NO.2所示，CL7-TteAgo融合蛋白的多核苷酸序列如SEQ ID NO.4所示。
用含有10mM咪唑的BufferA洗10个柱体积(分3次加入)，然后用含有200mM咪唑洗5个柱体积，取样进行SDS-PAGE检测。收集200mM咪唑的洗脱液(含有较高纯度目的蛋白)，与活化的偶联有Im7蛋白的琼脂糖珠孵育，融合表达的TteAgo-CL7融合蛋白会与Im7蛋白特异性结合，将融合蛋白特异性结合在琼脂糖珠上，通过高盐(1M NaCl)和低盐(100mM NaCl)反复洗脱(10次)除去杂蛋白，然后用3C蛋白酶进行切割获得纯的目的蛋白，收集纯化的蛋白并用SDS-聚丙烯酰胺凝胶鉴定纯度并超滤换液至Buffer B(20mM Tris–HCl pH 7.4、500mM NaCl、1mM TCEP)。将蛋白分成小份，经液氮速冻后，储存在-80℃。
图2显示了催化DEDX四联体所在的区域，以及TteAgo和其他Ago的序列一致性。图3显示了经过Ni-NTA柱和和分子筛对TteAgo进行纯化之后，凝胶分析TteAgo的结果。通过http://www.expasy.org/计算，TteAgo的预期大小约为118kDa。
实施例2TteAgo的切割活性
为了评估TteAgo能够裂解RNA/DNA向导和RNA/DNA靶的哪些组合，本实施例对所有可能的组合进行了活性测定。
裂解试验均在37℃下以5:2:1(TteAgo:向导:靶)摩尔比进行。将1uM TteAgo与400nM向导放在含有10mM HEPES-NaOH(pH 7.5)、100mM NaCl，5mM MnCl 2和5％甘油的反应缓冲液中混合，并在37℃孵育10分钟以用于向导加载。添加核酸靶至终浓度为200nM。37℃反应1h后，通过将样品与2x RNA上样染料(95％甲酰胺、18mM EDTA、0.025％SDS和0.025％溴酚蓝)混合并在95℃加热5分钟来终止反应。裂解产物通过20％变性TBE-PAGE解析，用SYBR Gold(Invitrogen)染色，用Gel Doc TM XR+(Bio-Rad)可视化。
图4中的A、B两幅图是用于测试的RNA向导、DNA向导、靶RNA、靶DNA的示意图，箭头表示预测的切割位点。图4中C、D图为TteAgo切割靶RNA和靶DNA的产物的尿素/聚丙烯酰胺凝胶电泳图，从图中可知：①在没有TteAgo的情况下进行孵育的DNA/RNA(引导/靶)对照测定中未观察到产物带(34nt)，表明产物带的形成是TteAgo核酸酶活性的结果；②TteAgo可以利用5’磷酸化的RNA向导、5’羟基化的RNA向导、5’磷酸化的DNA向导和5’羟基化的DNA向导切割靶RNA；③TteAgo可以利用5’磷酸化的RNA向导和5’羟基化的RNA向导切割靶DNA。
另外，对TteAgo催化四联体DEDD的第1个和第3个氨基酸D突变为氨基酸A，该双突变体DM记为TteAgo-DM，从图4C和4D可知，TteAgo-DM失去了DNA向导切割靶RNA和靶DNA的活性。
实施例3向导分子长度对切割效果的影响
参照实施例2的实验方法，采用不同长度的RNA向导或DNA向导与TteAgo结合，验证其切割靶RNA或靶DNA的活性。
检测结果如图5所示，图5中A图为12～30nt长度的5’磷酸化RNA向导条件下，30min内TteAgo表现出向导引导的靶RNA裂解的情况；图5中B图为12～30nt长度的5’磷酸化DNA向导条件下，30min内TteAgo表现出向导引导的靶RNA裂解的情况；图5中C图为12～30nt长度的5’磷酸化RNA向导条件下，60min内TteAgo表现出向导引导的靶ssDNA裂解的情况。从图5可知，RNA向导长度的范围为12-25nt、DNA向导长度的范围为12-30nt时可以有效的切割靶RNA；RNA向导长度的范围为12-30nt时可以有效的切割靶ssDNA。
实施例4金属离子对切割效果的影响
(1)金属离子种类的影响
参照实施例2的实验方法，在反应缓冲液中采用不同二价金属离子，包括Fe 2+，Co 2+，Ni 2+，Cu 2+，Zn 2+，Mg 2+，Mn 2+，Ca 2+等，验证阳离子对切割靶RNA或靶DNA的活性的影响。
检测结果如图6所示，可知阳离子为Mn 2+、Mg 2+、Co 2+或/和Ni 2+时，TteAgo结合RNA向导或DNA向导可以有效切割靶RNA(图6A、6B、6C、6D)；阳离子为Mn 2+条件下，TteAgo结合RNA向导可以有效切割靶DNA(图6E、6F)。
(2)金属离子浓度的影响
选取Mn 2+和Mg 2+，寻找15min内TteAgo表现出向导引导的靶RNA裂解的Mn 2+或Mg 2+浓度范围。
检测结果如图7所示：Mn 2+和Mg 2+浓度范围设置为0～10mM，向导为5’磷酸化的RNA时，Mn 2+浓度和Mg 2+浓度在0.05mM至10mM范围内时，都可以高效切割靶RNA(图7A和7B)；Mn 2+和Mg 2+浓度范围设置为0～10mM，向导为5’磷酸化的DNA时，Mn 2+浓度在2.5mM至10mM范围内时，而Mg浓度在 1mM至10mM范围内时，可以切割靶RNA(图7C和7D)；而Mn 2+浓度在1mM至50mM范围内，向导为5’磷酸化的RNA时，可以有效切割靶DNA(图7E和7F)。
实施例5温度对切割效果的影响
参照实施例2的实验方法，寻找15min内TteAgo表现出向导引导的靶RNA裂解的温度范围。结果如图8B和8D所示，5’磷酸化的RNA向导、5’羟基化的RNA向导在温度为25～70℃时可以切割靶RNA，其中37～60℃最好。
采用同样的方法，寻找60min内TteAgo表现出向导引导的靶DNA裂解的温度范围。结果如8A和8C所示，5’磷酸化的RNA向导、5’羟基化的RNA向导在温度为30～60℃时可以切割靶DNA，其中37～45℃最好。
实施例6
参照实施例2的实验方法，探究向导分子单碱基或双碱基突变对TteAgo切割靶RNA或靶DNA的影响，具体如下：
(1)RNA向导单碱基或/和双碱基突变对TteAgo切割靶RNA的影响
合成单个碱基或者双碱基突变的RNA向导(m1,m2,m3,m4,m5,m6,m7,
m8,m9,m10,m11,m12,m13,m14,m15,m16,m17,m18,m7m8,m8m9,m9m10,m10m11,m11m12,m12m13,m13m14，具体突变情况如图9所示)，上述RNA向导分别介导TteAgo切割靶RNA，结果如图10中A图所示：当RNA向导第11位和第12位碱基同时突变时，TteAgo对靶RNA的切割活性最弱。
(2)DNA向导单碱基突变对TteAgo切割靶RNA的影响
合成单个碱基突变的DNA向导(m1,m2,m3,m4,m5,m6,m7,m8,m9,m10,m11,m12,m13,m14,m15,m16,m17,m18，具体突变情况如图9所示)，上述DNA向导分别介导TteAgo切割靶RNA，结果如图10中B图所示：当DNA向导第8位、第9位、第11位或第12位碱基突变时，TteAgo对靶RNA的切割活性最弱。
(3)RNA向导单碱基突变对TteAgo切割靶DNA的影响
分别合成单个碱基突变的RNA向导(m1,m2,m3,m4,m5,m6,m7,m8,m9,m10, m11,m12,m13,m14,m15,m16,m17,m18，具体突变情况如图9所示)，上述RNA向导分别介导TteAgo切割靶DNA，结果如图10中C图所示：当RNA向导第3位至第17位出现单个碱基突变时，TteAgo对靶DNA的切割活性显著减弱。
综上所述，本发明提供的真核生物来源的Argonaute蛋白对RNA向导和ssDNA向导有结合活性，同时具有对靶RNA和靶DNA的均具有核酸酶活性，而且本发明的eAgo蛋白能够对细胞内和细胞外的遗传物质进行位点特异性修饰。故其可有效地应用于生物技术的许多领域，比如核酸检测、基因编辑和基因修饰等。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，都应涵盖在本发明的保护范围之内。

Claims (6)

  1. 一种eAgo复合物在细胞内或体外特异性切割靶核酸中的应用，其特征在于，所述eAgo复合物由Argonaute蛋白与向导分子复合形成；
    所述Argonaute蛋白具有核酸酶活性，且所述Argonaute蛋白为如下任一所示蛋白：
    A1).氨基酸序列如SEQ ID NO.1所示的蛋白，
    A2).与SEQ ID NO.1所示的氨基酸序列具有至少50％的序列一致性且具有相同功能的蛋白；
    编码所述Argonaute蛋白的核酸分子为如下任一所示：
    B1).核苷酸序列如SEQ ID NO.3所示的DNA分子，
    B2).在严格条件下与SEQ ID NO.3所示的DNA分子杂交且编码权利要求1所述Argonaute蛋白的DNA分子，
    B3).与B1)或B2)限定的DNA序列具有至少50％序列一致性且编码权利要求1所述Argonaute蛋白的DNA分子；
    所述向导分子为ssDNA向导或RNA向导，所述向导分子为5’末端磷酸化的RNA向导、5’末端羟基化的RNA向导、5’末端磷酸化的ssDNA向导或5’末端羟基化的ssDNA向导，且所述ssDNA向导的长度为12至30个核苷酸。
  2. 根据权利要求1所述的应用，其特征在于，所述eAgo复合物在体内或体外特异性切割靶核酸的方法具体为：令所述eAgo复合物与靶核酸接触，所述靶核酸含有与所述向导分子至少12个碱基互补的核苷酸序列，所述eAgo复合物特异性切割靶核酸。
  3. 根据权利要求1所述的应用，其特征在于，所述Argonaute蛋白在10～65℃具有核酸酶活性。
  4. 根据权利要求1所述的应用，其特征在于，所述Argonaute蛋白在二价金属阳离子的溶液中具有核酸酶活性，所述二价金属阳离子为Fe 2+、Co 2+、Ni 2+、 Cu 2+、Zn 2+、Mg 2+、Mn 2+、Ca 2+中的至少一种。
  5. 根据权利要求4所述的应用，其特征在于，所述二价金属阳离子为Mn 2+和/或Mg 2+。
  6. 根据权利要求1所述的应用，其特征在于，所述Argonaute蛋白的核酸酶活性具有单碱基和/或双碱基专一性。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105483118A (zh) * 2015-12-21 2016-04-13 浙江大学 以Argonaute核酸酶为核心的基因编辑技术
US20170367280A1 (en) * 2016-05-27 2017-12-28 Kws Saat Se Use of argonaute endonucleases for eukaryotic genome engineering
CN110229799A (zh) * 2018-03-06 2019-09-13 北京贝瑞和康生物技术有限公司 Argonaute蛋白突变体及其用途
CN112538470A (zh) * 2020-12-11 2021-03-23 湖北大学 一种原核生物来源的Argonaute蛋白及其应用
CN114606215A (zh) * 2022-01-24 2022-06-10 湖北大学 一种真核生物来源的Argonaute蛋白及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108796036B (zh) * 2018-04-03 2021-11-19 交弘生物科技(上海)有限公司 基于原核Argonaute蛋白的核酸检测方法及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105483118A (zh) * 2015-12-21 2016-04-13 浙江大学 以Argonaute核酸酶为核心的基因编辑技术
US20170367280A1 (en) * 2016-05-27 2017-12-28 Kws Saat Se Use of argonaute endonucleases for eukaryotic genome engineering
CN110229799A (zh) * 2018-03-06 2019-09-13 北京贝瑞和康生物技术有限公司 Argonaute蛋白突变体及其用途
CN112538470A (zh) * 2020-12-11 2021-03-23 湖北大学 一种原核生物来源的Argonaute蛋白及其应用
CN114606215A (zh) * 2022-01-24 2022-06-10 湖北大学 一种真核生物来源的Argonaute蛋白及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 2 September 2014 (2014-09-02), ANONYMOUS : "hypothetical protein MYCTH_2306810 [Thermothelomyces thermophilus ATCC 42464]", XP093079425, retrieved from Genbank Database accession no. AEO58979 *
DATABASE Protein 27 November 2019 (2019-11-27), ANONYMOUS: "uncharacterized protein MYCTH_2306810 [Thermothelomyces thermophilus ATCC 42464]", XP093079419, retrieved from Genbank Database accession no. XP_003664224 *

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