High-temperature prokaryotic argonaute nuclease having both DNA and RNA cleavage activity and use thereof

Abstract

The present invention belongs to the field of synthetic biology and biotechnology. Disclosed are a high-temperature prokaryotic Argonaute nuclease having both DNA and RNA cleavage activity and the use thereof. The thermophilic-bacterium-derived Argonaute nuclease is specifically A1) or A2) below: A1) a protein having an amino acid sequence as shown in SEQ ID NO: 1; and A2) a fusion protein obtained by means of linking a tag to the N-terminus or / and C-terminus of A1). The enzyme can mediate the cleavage of a single-stranded nucleic acid target by using both 5'-phosphorylated gDNA and 5'-hydroxylated gDNA; is resistant to high temperatures of 50-85°C, and has high specificity; and is conducive to advancing the development of tools for DNA and RNA manipulation in vivo and in vitro.

Description

High-temperature prokaryotic argonaute nuclease with DNA and RNA cleavage and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology and biotechnology, and in particular to a high-temperature prokaryotic Argonaute nuclease with DNA and RNA cleavage and application thereof. BACKGROUND

[0002] Argonaute (Ago) was first discovered by Karen Bohmert in 1998 in a study describing Arabidopsis thaliana mutants. Since the leaves of the mutant were curled like octopus tentacles, the gene and the corresponding protein were named after Argonauta argo. Later, as research deepened, it was found that Ago protein is a programmable nuclease widely present in eukaryotes and prokaryotes, and is highly conserved in evolution. According to its source, Agos can be divided into pAgos and eAgos, most of which have high structural homology. Catalytically active pAgos and eAgos have similar catalytic processes, including guide binding, target recognition and catalysis. It is generally believed that eAgos is evolved from pAgos, but most of the characterized pAgos cannot cut target RNA under the guidance of gRNA as eAgos, and the physiological function of pAgos in vivo is also not as clear as eAgos.

[0003] Compared with Cas nucleases widely used in gene editing, Ago has no PAM (Protospacer adjacent motif) and PFS (Protospacer flanking site) requirements when recognizing targets, has a wider targeting range, and uses guide nucleic acids that can be DNA and RNA, generally shorter in length. Compared with normal temperature Ago, the application prospect of high temperature Ago is more extensive, because many normal temperature Agos do not have unwinding function and cannot open DNA double strands, which limits the application of Ago. However, under high temperature conditions, DNA can open double strands by itself, which allows Ago protein to play a role as a nuclease. Therefore, there is a nucleic acid detection technology.

[0004] At present, only a few pAgos have been characterized, and the in vitro application of pAgos mainly uses PfAgo and TtAgo from thermophilic organisms. Therefore, in order to increase the understanding of pAgos and better promote the development of in vitro gene editing tools, it is crucial to explore new high-temperature pAgos and their in vitro nuclease properties.

[0005] SUMMARY

[0006] The present application aims at providing a high-temperature prokaryotic Argonaute nuclease with the ability of cutting both DNA and RNA and its application, which has high-temperature thermal stability and can efficiently and specifically cut DNA or RNA.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The first purpose of the present application is to provide a high-temperature prokaryotic Argonaute nuclease with the ability of cutting both DNA and RNA, which is derived from thermophilic bacteria, and the Argonaute nuclease is specifically as follows A1) or A2):

[0009] A1) the amino acid sequence is shown as SEQ ID NO. 1;

[0010] A2) a fusion protein obtained by connecting a tag to the N terminal or / and C terminal of A1).

[0011] In order to facilitate the purification of the protein in A1), the following tags can be connected to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown as SEQ ID No. 1 in the sequence listing: Poly-Arg (RRRRR), Poly-His (HHHHHH), FLAG (DYKDDDK).

[0012] The second purpose of the present application is to provide biological materials related to the above-mentioned Argonaute nuclease, which are any of the following B1) to B6): B1) a nucleic acid molecule encoding the Argonaute nuclease of claim 1; B2) an expression cassette containing the nucleic acid molecule of B1);

[0013] B3) a recombinant vector containing the nucleic acid molecule of B1);

[0014] B4) a recombinant microorganism containing the nucleic acid molecule of B1);

[0015] B5) a recombinant microorganism containing the expression cassette of B2);

[0016] B6) a recombinant microorganism containing the recombinant vector of B3).

[0017] The third purpose of the present application is to provide the use of the above-mentioned biological materials in the preparation of Argonaute nuclease.

[0018] The fourth purpose of the present application is to provide a preparation method of Argonaute nuclease, which comprises the step of expressing the coding gene of the above-mentioned protein in a biological cell to obtain a protein with Argonaute nuclease activity; and the biological cell is a microbial cell.

[0019] A fifth object of the present application is to provide the use of the above-mentioned Argonaute nuclease or biological material in cleaving nucleic acid.

[0020] A sixth object of the present application is to provide a nucleic acid cleavage system comprising the above-mentioned Argonaute nuclease, guide DNA, target nucleic acid and reaction buffer.

[0021] Further, the length of the guide DNA is 13-40 nt, and the 5' end of the guide DNA is phosphorylated.

[0022] Preferably, the length of the guide DNA is 15-30 nt, such as 18, 19 or 20 nt.

[0023] Further, the specificity of the Argonaute nuclease is that the guide DNA is complementary to the target nucleic acid at 10-15 positions.

[0024] Further, it further comprises Mn 2+ or Mg 2+ , the concentration of the Mn 2+ is 0.05-10 mM, and the concentration of the Mg 2+ is 0.5-10 mM.

[0025] Further, the temperature of the nucleic acid cleavage system is 30-85℃.

[0026] Further, the concentration of NaCl in the reaction buffer is 25-250 mM.

[0027] Further, the target nucleic acid is one of single-stranded DNA, double-stranded DNA or plasmid DNA.

[0028] A seventh object of the present application is to provide the use of the above-mentioned Argonaute nuclease or the above-mentioned nucleic acid cleavage system in preparing a reagent or kit for DNA molecular manipulation.

[0029] An eighth object of the present application is to provide the use of the above-mentioned Argonaute nuclease or the above-mentioned nucleic acid cleavage system in preparing a reagent or kit or biosensor for detecting target nucleic acid molecules.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] (1) The present application provides a high-temperature prokaryotic Argonaute nuclease capable of cleaving both DNA and RNA, which is derived from thermophilic bacteria, and the enzyme can mediate high-efficiency cleavage of single-stranded nucleic acid targets using 5'-phosphorylated gDNA.

[0032] (2) The Argonaute nuclease provided by the application has an optimum reaction temperature range of 50-85℃; can utilize Mn 2+ , Mg 2+ as active ions, maintains high activity, has certain tolerance to NaCl concentration, and the tolerance range is between 25-250mM; has no obvious preference to the first base of the 5' end of gDNA; the enzyme can cut single-stranded nucleic acid targets, double-stranded linear DNA and plasmid DNA, and can cut supercoiled DNA into linear DNA at a position with a GC content of 29%-65% of the plasmid, which enables the enzyme to be used for related molecular operations of DNA, such as DNA molecular cloning. The enzyme has good DNA and RNA cutting activity and high specificity, and has a wide application prospect in the field of nucleic acid detection. GgeAgo will facilitate the development of in vivo and in vitro DNA and RNA operation tools. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is the result of phylogenetic analysis (A) and multiple sequence alignment (B) of GgeAgo;

[0034] Figure 2 is the result of SDS PAGE electrophoresis analysis of the purified GgeAgo protein;

[0035] Figure 3 is the result of the determination of the cutting activity of GgeAgo;

[0036] Figure 4 is the effect of the length of 5' phosphorylated gDNA (A) on the cutting activity of GgeAgo;

[0037] Figure 5 is the result of the effect of the type of divalent metal ion on the cutting activity of GgeAgo;

[0038] Figure 6 is the result of the effect of the concentration of Mn 2+ (A) and Mg 2+ on the cutting activity of GgeAgo;

[0039] Figure 7 is a result graph of the temperature range required for the reaction of GgeAgo;

[0040] Figure 8 is the result of the NaCl concentration range that can be tolerated by GgeAgo;

[0041] Figure 9 is the result of the preference of GgeAgo to the first base of the 5' end of gDNA;

[0042] Figure 10 is the result of the cutting discrimination of GgeAgo to single-point mismatches between gDNA and Target at different positions;

[0043] Figure 11 shows the results of GgeAgo cleavage of double-stranded linear DNA (A) and cleavage of pUC19 plasmid 29%-65% GC content positions. OC represents open circular plasmid (one strand of the plasmid is broken); LIN represents linearized plasmid (both strands of the plasmid are broken); SC represents supercoiled plasmid. DETAILED DESCRIPTION

[0044] In order to make the objects, technical solutions and advantages of the present application clearer, the embodiments of the present application are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0045] In the following examples, the experimental methods used are conventional methods, and are generally performed according to conventional conditions such as the conditions described in Molecular Cloning Experiments Guide (Fourth Edition) (Chinese Edition) published by Science Press, or according to the conditions recommended by the manufacturer, which are well-known experimental operations to those skilled in the art, and the present application does not limit this.

[0046] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0047] The present application mines a high-temperature-resistant Argonaute nuclease, referred to as GgeAgo, from a thermophilic bacterium as a starting strain. The present application constructs a recombinant plasmid pET28a-GgeAgo by PCR technology, and realizes the heterologous expression of GgeAgo by transforming the recombinant plasmid into Escherichia coli, and then purifies the protein GgeAgo produced by the recombinant strain through a Ni NTA column.

[0048] The nucleic acid cleavage system provided by the present application comprises:

[0049] (a) a guide DNA (gDNA);

[0050] (b) an Argonaute (Ago) nuclease;

[0051] (c) a target nucleic acid, a single-stranded DNA (ssDNA), a single-stranded RNA (ssRNA), a double-stranded DNA (dsDNA), and a plasmid DNA.

[0052] Example 1

[0053] Heterologous expression and purification of Argonaute nuclease:

[0054] The nucleotide sequence of GgeAgo was synthesized by Jin Kai Rui Biological Technology Co., Ltd., and was cloned into pET28a expression vector with a C-terminal 6xHis tag to obtain pET28a-GgeAgo plasmid, which was then transformed into E. coli Rosetta (DE3). A single colony was inoculated into LB liquid medium containing kanamycin, and cultured at 37°C in a shaker. When the OD 600 When the OD of the bacteria reached 0.6-0.8, the culture was moved to a shaker at 18°C, and IPTG was added for overnight induction. The bacteria were collected by centrifugation, washed with buffer, resuspended in Buffer A, and added with PMSF at a final concentration of 1 mM. The bacteria were broken by high pressure. The supernatant was collected by centrifugation. After filtering the supernatant, Ni-NTA purification was performed. Gradient elution was performed with 20 mM-500 mM imidazole, and samples were taken for SDS-PAGE detection. The elution fraction containing the high-purity target protein was collected. The purified protein was analyzed by SDS-PAGE to determine the purity, and the elution fraction containing the high-purity target protein was collected and stored for later use.

[0055] The results of SDS-PAGE identification and analysis of the high-purity GgeAgo protein obtained by final purification are shown in Figure 2. The protein band of GgeAgo is consistent with the expected size of 82.3 kDa calculated by http: / / www.expasy.org / . The phylogenetic tree of the GgeAgo and some characterized Argonaute proteins (Ago proteins) is shown in Figure 1A. The catalytic DEX tetrad of GgeAgo and the sequence alignment with seventeen characterized Ago proteins are shown in Figure 1B.

[0056] Example 2

[0057] Determination of Argonaute nuclease cleavage activity:

[0058] A 45 nt single-stranded DNA, RNA target nucleic acid and four complementary 18 nt guide DNA, RNA were designed and synthesized by Bio Company, and the specific sequences are shown in Figure 3 (top). The reaction buffer (containing 10 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 5% glycerol) was prepared, and the final concentration of MnCl2 was 5 mM, 800 nM GgeAgo, 400 nM synthetic gDNA or gRNA was added to the reaction buffer, and the Ago-gDNA complex was incubated at 50°C for 10 min, then 200 nM fluorescent single-stranded DNA or RNA target nucleic acid complementary to the guide sequence was added, and the reaction was carried out at 50°C for 30 min. After the reaction, the loading buffer (containing 95% formamide, 18 mM EDTA, 0.025% bromophenol blue, 0.025% SDS) was added at a ratio of 1:1, mixed and heated at 95°C for 5 min to terminate the reaction. The cleavage of GgeAgo on single-stranded nucleic acid target was detected by 20% denaturing PAGE electrophoresis, stained with SYBR Gold (Invitrogen) and visualized by Gel DocTM XR+ (Bio-Rad), and the results are shown in Figure 3 (bottom).

[0059] The results show that no product band (34 nt) is observed in the control assay of guide and target reaction (DNA / RNA) without GgeAgo, indicating that the formation of the product band is the result of GgeAgo nuclease activity. GgeAgo can cleave DNA and RNA targets using 5' phosphorylated guide DNA and 5' hydroxylated guide DNA, and the cleavage activity of 5' phosphorylated guide is higher.

[0060] Example 3

[0061] Determination of Argonaute nuclease cleavage activity:

[0062] 1. Investigation of the effect of different lengths of guide DNA on the recognition and cleavage of single-stranded nucleic acid target by GgeAgo.

[0063] 5' phosphorylated DNA with a length of 13-40 nt was selected as guide DNA, which was incubated with GgeAgo to form a complex, and the effect of different lengths of guide DNA on the recognition and cleavage of single-stranded nucleic acid target by GgeAgo was determined. The determination results are shown in Figure 4.

[0064] The results show that the length of the guide DNA has a certain effect on the activity of GgeAgo recognizing and cleaving single-stranded nucleic acid target, and GgeAgo can effectively cleave single-stranded nucleic acid target using guide DNA with a length of 15-30 nt, and the cleavage effect is best when the length of the guide DNA is in the range of 18-30 nt.

[0065] 2. Investigate the effect of divalent metal cations on the cleavage activity of Argonaute nuclease.

[0066] To investigate the effect of divalent metal cations on the cleavage activity of GgeAgo, GgeAgo and guide DNA were mixed in a reaction buffer containing different divalent metal cations at a final concentration of 5 mM, and incubated at 50°C for 10 min to form an Ago-gDNA complex, and then nucleic acid targets were added for cleavage activity detection. The divalent metal cations are selected from Mn 2+ , Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Co 2+ , Zn 2+ and Ni 2+ , to determine the effect of different metal cations on cleavage activity, and the determination results are shown in Figure 5.

[0067] The results show that GgeAgo can use Mn 2+ , Mg 2+ , Co 2+ as metal ions to mediate 5' phosphorylated guide DNA guided single-stranded DNA target cleavage, and can also use Mn 2+ , Mg 2+ as metal ions to mediate 5' phosphorylated guide DNA guided single-stranded RNA target cleavage. Among them, GgeAgo prefers Mn 2+ .

[0068] Further explore the effect of Mn 2+ and Mg 2+ concentration on the cleavage activity of GgeAgo, select 0.01 mM to 10.00 mM of Mn 2+ or 0.5 mM to 10.00 mM of Mg 2+ added to the buffer, and determine the cleavage activity of GgeAgo on single-stranded nucleic acid targets, and the determination results are shown in Figure 6.

[0069] The results show that the concentration of divalent metal cations has a certain effect on the cleavage activity of GgeAgo. When the final concentration of Mn 2+ in the reaction system is ≥0.05 mM or the final concentration of Mg 2+ is ≥2.5 mM, obvious cleavage products can be produced.

[0070] 3. Investigate the effect of temperature on the cleavage activity of Argonaute nuclease.

[0071] After incubation of GgeAgo with guide DNA to form complex, the target sequence was added and reacted for 15 min at different temperatures (30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C), and the cleavage activity was determined. The results are shown in Figure 7.

[0072] The results show that when the guide is 5' phosphorylated DNA, GgeAgo can cleave single-stranded nucleic acid targets at a temperature range of 30-85°C, and the activity increases with increasing temperature. It is worth noting that because the active temperature of RNase inhibitor is about 65°C, the reaction temperature above 65°C will cause obvious degradation of the RNA target, so the highest temperature for the cleavage experiment of the RNA target is only 75°C. In summary, GgeAgo can exert endonuclease activity under a wide range of temperature conditions.

[0073] 4. Investigate the effect of different reaction buffer concentrations on Argonaute nuclease cleavage activity.

[0074] Adjust the reaction buffer composition, and configure reaction buffer with a final concentration of 10 mM HEPES-NaOH pH 7.5 and different concentrations of NaCl (25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM), respectively. After adding the nucleic acid cleavage system and reacting for 15 min, the cleavage activity was determined. The results are shown in Figure 8.

[0075] The results show that GgeAgo can exert cleavage activity at a NaCl final concentration of 25-50 mM. Among them, the GgeAgo cleavage activity is best when the final concentration of NaCl is 100 mM.

[0076] 5. Investigate the cleavage discrimination of GgeAgo to single-base non-complementary between guide DNA and target nucleic acid.

[0077] Design 18nt gDNA with the first base at the 5' end of gDNA being A, T, G, and C, respectively, and add it to the corresponding nucleic acid cleavage system. After reacting for 5-60 min, respectively, the preference of GgeAgo for the first base at the 5' end of gDNA was determined under the mediation of 5' phosphorylated guide DNA.

[0078] The results are shown in Figure 9. The results show that GgeAgo has no obvious preference for the first base at the 5' end of gDNA, and can be any of the four bases.

[0079] A series of gDNAs with single-point mismatches at different sites (MP 1-18) with single-stranded nucleic acid targets were designed, and the remaining reaction conditions were unchanged. The single-stranded nucleic acid cleavage system was added and reacted, and the discrimination cleavage effect of GgeAgo was determined.

[0080] The results are shown in Figure 10. The results show that when the mismatch occurs at positions 7, 10-15 of the gDNA, the activity of GgeAgo in cleaving the DNA target is significantly reduced, and the lowest cleavage activity occurs at position 11 of the gDNA; and when the mismatch occurs at positions 11-15 of the gDNA, the activity of GgeAgo in cleaving the RNA target is significantly reduced, and the lowest cleavage activity occurs at position 12 of the gDNA.

[0081] Example 4

[0082] Research on Argonaute nuclease cleavage of DNA molecules:

[0083] Forward and reverse gDNAs targeting GC contents of 29%, 39%, 45%, 53%, 64%, and 65% (within 200 bp upstream and downstream of the cleavage site) were designed and synthesized for cleaving double-stranded DNA and two strands of plasmid pUC19, and the sequences are shown in Table 1.

[0084] Table 1.

[0085] After GgeAgo was incubated with the corresponding forward and reverse gDNAs of different GC contents of the target cleavage site to form Ago-single-stranded gDNA complexes, the complexes formed by the forward and reverse gDNAs with GgeAgo were mixed, and 200 ng of double-stranded DNA target or pUC19 plasmid was added and reacted at 70°C for 30 min. After the reaction, 100 ng of the reaction sample was added to 5x loading buffer, and electrophoresis was performed in 1% agarose gel for detection.

[0086] The results are shown in Figure 11. The results show that GgeAgo can effectively cleave double-stranded DNA with GC contents of 29-64% near the cleavage site; when the GC content near the cleavage site is 29% and 39%, GgeAgo can use a pair of gDNAs to completely cleave the supercoiled plasmid into linear plasmid; when the GC content near the cleavage site is 45%, 53%, 64%, and 65%, GgeAgo can use a pair of gDNAs to convert the supercoiled plasmid into open circle plasmid and linear plasmid.

[0087] The above not involved, applicable to the prior art.

[0088] Although some specific embodiments of the present application have been described in detail by way of example with reference to the drawings, it is to be understood that the above examples are intended to be illustrative only and are not intended to limit the scope of the present application, and that various modifications and changes can be made by those skilled in the art to the particular embodiments described without departing from the spirit and scope of the present application. It is intended that the scope of the present application be limited only by the broadest interpretation of the appended claims to be accorded under 35 U.S.C. § 112.

Claims

1. A high-temperature prokaryotic Argonaute nuclease with simultaneous cleavage of DNA and RNA, characterized in that, The Argonaute nuclease derived from thermophilic bacteria is specifically as follows A1) or A2): A1) the amino acid sequence shown in SEQ ID NO. 1; A2) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of A1).

2. A biological material related to the Argonaute nuclease of claim 1, which is any one of the following B1) to B6): B1) a nucleic acid molecule encoding the Argonaute nuclease of claim 1; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1); B4) a recombinant microorganism containing the nucleic acid molecule of B1); B5) a recombinant microorganism containing the expression cassette of B2); B6) a recombinant microorganism containing the recombinant vector of B3).

3. Use of the biological material of claim 2 in the preparation of an Argonaute nuclease.

4. A method of producing an Argonaute nuclease, characterized in that, The method comprises the step of expressing the gene encoding the Argonaute nuclease of claim 1 in a biological cell to obtain a protein having Argonaute nuclease activity; and the biological cell is a microbial cell.

5. Use of the Argonaute nuclease of claim 1 or the biological material of claim 2 in nucleic acid cleavage.

6. A nucleic acid cleaving system, characterized by, The nucleic acid cleavage system can be used in vivo and in vitro, which comprises: (a) a guide DNA; (b) the Argonaute nuclease of claim 1; (c) an optional target nucleic acid; (d) a reaction buffer.

7. The nucleic acid cleaving system of claim 6, wherein, The length of the guide DNA is 13-40 nt, the 5' end of the guide DNA is phosphorylated and can be any of the four bases.

8. The specificity of the Argonaute nuclease of claim 6, characterized in that, The guide DNA and the target nucleic acid have complementarity at positions 10-15.

9. The nucleic acid cleaving system of claim 6, wherein, including Mn 2+ or Mg 2+ , the concentration of said Mn 2+ is 0.05-10 mM, and the concentration of said Mg 2+ is 0.5-10 mM.

10. The nucleic acid cleaving system of claim 6, wherein The temperature of the nucleic acid cleavage system is 30-85℃, and the concentration of NaCl in the reaction buffer of the nucleic acid cleavage system is 25-250 mM.

11. The nucleic acid cleaving system of claim 6, wherein, The target nucleic acid is one of single-stranded RNA, single-stranded DNA, double-stranded DNA or plasmid DNA.

12. Use of the Argonaute nuclease of claim 1 or the nucleic acid cleavage system of any one of claims 6-11 in the preparation of a reagent or kit for DNA molecular manipulation.

13. Use of the Argonaute nuclease of claim 1 or the nucleic acid cleavage system of any one of claims 6-11 in the preparation of a reagent or kit or biosensor for detecting a target nucleic acid molecule.

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