Modified pae casdin g helicase and applications thereof

Abstract

The application provides a modified Pae CasDinG helicase and application thereof, and relates to the technical field of gene sequencing. The application firstly provides a CasDinG helicase truncated body with helicase activity, and further provides a modified Pae CasDinG helicase in which a WalkerA motif and a WalkerB motif in an HD1 domain are covalently connected through a connecting molecule, and the modification improves the stability of the Pae CasDinG helicase in the application in polynucleotide binding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene sequencing, molecular detection and clinical detection, and in particular to a modified PaeCas DinG helicase and application thereof. BACKGROUND

[0002] CasDinG is a key component in the CRISPR-Cas acquired immunity type IV-A system of bacteria for resisting foreign invading genetic factors, which has 5'-3' DNA helicase activity dependent on adenine nucleotide triphosphate (ATP) and metal ions, and in order to further develop the sequencing application of the protein, the present application is proposed. SUMMARY

[0003] The present application first provides a truncated Pae CasDinG helicase, which is a Pae CasDinG helicase (PDB:8e2w) protein removing an N-terminal domain, and the sequence is shown in SEQ ID NO:1.

[0004] In another aspect of the present application, a modified Pae CasDinG helicase is provided, wherein the parent sequence of the modified Pae CasDinG helicase is SEQ ID NO:1 or a sequence having 90% homology with SEQ ID NO:1; and the modified Pae CasDinG has at least one covalent linkage between 164-171 and 326-344, or between 354-361 and 492-495.

[0005] Preferably, the parent sequence of the modified Pae CasDinG helicase has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with SEQ ID NO:1.

[0006] Preferably, the covalent linkage exists between at least one amino acid residue at positions 164-171 and at least one amino acid residue at positions 326-344, or between at least one amino acid residue at positions 354-361 and at least one amino acid residue at positions 492-495, with reference to SEQ ID NO:1.

[0007] In the detailed description of the present application, the amino acid residues with covalent linkage are preferably at least one of E167, S166, D168, and at least one of R327, D328, R330, S331, or a combination of P356 and K493.

[0008] In the detailed description of the application, the covalent linkage is achieved by replacing or inserting cysteine or unnatural amino acids.

[0009] In the detailed description of the application, at least one cysteine is introduced at each of positions 164-171 and 326-344, or at least one cysteine is introduced at positions 354-361 and 492-495; further preferably, at least one of E167C, S166C, D168C and at least one of R327C, D328C, R330C, S331C or a combination of P356C and K493C are introduced; more preferably, a combination of E167C and R327C is introduced.

[0010] In the detailed description of the application, the covalent linkage between two cysteines is achieved by a disulfide bond or linker molecules; preferably, the linker molecules include BMOE (Bismaleimidoethane), BMB (1,4-Bismaleimidotbutane), BMH (Bismaleimido-hexane), DTME (Dithiomaleimidoethane), TMEA (Tris(2-bismaleimidoethyl)amine), Bis(PEG)2 (1,8-Bismaleimido-diglycol) and Bis(PEG)3 (1,11-Bismaleimido-triglycol).

[0011] Preferably, the covalent linkage is achieved by bismaleimidoethane, Bis(PEG)2 or Bis(PEG)3; further preferably, the covalent linkage is achieved by bismaleimidoethane.

[0012] In another detailed description of the application, the covalent linkage is achieved by introducing at least one unnatural amino acid.

[0013] Preferably, the covalent linkage is achieved by a bio-orthogonal reaction involving unnatural amino acids; more preferably, the covalent linkage is achieved by a bio-orthogonal reaction involving linker molecules.

[0014] Preferably, the bio-orthogonal reaction includes condensation reaction between ketone carbonyl compounds and hydroxylamine / hydrazine, 1,3-dipolar cycloaddition reaction, Staudinger ligation reaction, inverse electron-demand Diels-Alder reaction (IEDDA) reaction or photo-initiated cross-linking reaction.

[0015] Preferably, the unnatural amino acids include ketone / aldehyde group, oxime / hydroxylamine group, azido group, alkyne group, cyclophane group, tetrazine group or indolizine group.

[0016] Preferably, the modified Pae CasDinG helicase further comprises substitution of one or more cysteines in the wild-type sequence; the cysteine residue sites present in SEQ ID NO: 1 are: 144, 172, 193, 434, 448, 467, 500; preferably, 5-7 cysteines in SEQ ID NO: 1 are substituted.

[0017] Preferably, the modified Pae CasDinG helicase further comprises substitution of one or more cysteines in the wild-type sequence; the cysteine residue sites present in SEQ ID NO: 1 are: 144, 172, 193, 434, 448, 467, 500; preferably, 5-7 cysteines in SEQ ID NO: 1 are substituted.

[0018] Preferably, the substitution of the original cysteine is substitution with threonine and / or serine; preferably, the substitution of the original cysteine is substitution with the following amino acids: C144S, C172S, C434S, C448S and C500S.

[0019] In the specific embodiments of the present application, the modified Pae CasDinG helicase comprises the amino acid sequence shown in SEQ ID NO: 3, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with the amino acid sequence shown in SEQ ID NO: 3, and there is a covalent linkage between E167C and R327C.

[0020] The present application also provides a sequencing element comprising the truncated Pae CasDinG helicase or the modified Pae CasDinG helicase described above, and a sequencing device.

[0021] The present application also provides a sequencing use of the truncated Pae CasDinG helicase or the modified Pae CasDinG helicase described above.

[0022] In another aspect, the present application provides a mutant Pae CasDinG helicase, the parent sequence of which is SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1; and has at least one cysteine or unnatural amino acid at each of positions 164-171 and 326-344, or at least one cysteine or unnatural amino acid is introduced at positions 354-361 and 492-495; further preferably, the following are introduced: at least one of E167C, S166C, D168C and at least one of R327C, D328C, R330C, S331C or a combination of P356C and K493C; more preferably, a combination of E167C and R327C.

[0023] Preferably, in the present application, the mutant Pae CasDinG helicase further comprises substitution of the original one or more cysteines with an amino acid; the cysteine residue sites originally present in SEQ ID NO: 1 are: 144, 172, 193, 434, 448, 467, and 500. Preferably, 5-7 cysteines in SEQ ID NO: 1 are substituted.

[0024] Preferably, the mutant Pae CasDinG helicase further comprises substitution of the original cysteine with an amino acid; the substitution is with another natural amino acid or an unnatural amino acid; the other natural amino acid or unnatural amino acid does not contain a thiol group; preferably, the other 19 natural amino acids, including glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; more preferably, threonine and / or serine.

[0025] Preferably, the original cysteine is substituted with threonine and / or serine; preferably, the original cysteine is substituted with the following amino acids: C144S, C172S, C434S, C448S, and C500S.

[0026] Preferably, the mutant Pae CasDinG helicase comprises the amino acid sequence set forth in SEQ ID NO: 3, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with the amino acid sequence set forth in SEQ ID NO: 3.

[0027] The present application also provides a polynucleotide encoding the short Pae CasDinG helicase or the mutant Pae CasDinG helicase.

[0028] The present application also provides a biological material, such as a vector or a recombinant microorganism, comprising the polynucleotide or expressing the mutant Pae CasDinG helicase.

[0029] The present application also provides a method for preparing the modified Pae CasDinG helicase, comprising the following steps:

[0030] 1) providing the mutant Pae CasDinG helicase as described above;

[0031] 2) reacting to obtain the modified Pae CasDinG helicase under conditions for forming a covalent bond.

[0032] Preferably, the reaction of step 2) is a thiol-involved reaction.

[0033] Preferably, the reaction of step 2) is a bio-orthogonal reaction.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] The present application first provides a Pae CasDinG helicase truncated body having helicase activity, and further provides a modified Pae CasDinG helicase in which the Walker A motif and the Walker B motif in the HD1 domain are covalently connected by a linker molecule, which modification improves the stability of the Pae CasDinG helicase of the present application in binding to a polynucleotide, and especially when the length of the polynucleotide increases, the modified helicase of the present application can still stably control the movement of the polynucleotide without falling off the polynucleotide. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is an electropherogram of the Pea CasDinG mutant after modification by a linker molecule;

[0037] Band 1 represents the Pae CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S mutant; and band 2 represents the mutant after modification by a linker molecule.

[0038] Figure 2 shows the effect of the Pea CasDinG helicase on the DNA binding ability before and after modification;

[0039] Lane 1 represents a simple DNA substrate without protein, lane 2 represents the result of wild type Pae CasDinG binding to the DNA substrate at a molar ratio of 5:1, and lane 3 represents the result of modified Pae CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S binding to the substrate DNA at a molar ratio of 5:1;

[0040] Figure 3 is a schematic diagram of DNA construct X;

[0041] Figure 4 is a pore current graph of Pae CasDinG mutant capable of controlling the complete DNA construct X through the nanopore;

[0042] Figure 5 is an enlarged view of a partial region of Figure 4

[0043] Figure 6 is a pore signal graph of a simple nucleic acid sample without helicase control. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of the present application.

[0045] Technical terms

[0046] As used in the present application, "covalent linkage" refers to a linkage between two or more amino acid residues at specific positions of Pae CasDinG helicase through any chemical group, including direct linkage through functional groups of amino acid residues, or linkage through bifunctional linking molecules or multifunctional linking molecules.

[0047] For direct linkage, for example, a disulfide bond can be formed by two cysteines, or two amino acid residues carry biorthogonal reaction functional groups, such as carrying azide or alkyne respectively, to form a triazole linkage under the catalysis of Cu ions.

[0048] ​In case of covalent linkage using a bifunctional linking molecule, the “covalent linkage” has the typical structure of A-L-A1, wherein A and A1 are structures formed upon coupling of a functional group of the linker with a functional group of the amino acid residue, such as a thioether (e.g. formed from the reaction of a thiol of cysteine with a maleimide) or a triazole (formed from the reaction of an azide and an alkyne), and wherein L is the spacer of the bifunctional linker, which can be any divalent chemical group, preferably a C1-10 (including C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10 straight or branched alkylene group or a polyethylene glycol subunit of 1, 2 or 3 repeating units.

[0049] Bifunctional linking molecules are well known to the skilled person and are either directly commercially available or can be custom made. In some embodiments of the present application, homobifunctional linkers such as BMOE (Bis-Maleimidoethane) or BMB (1,4-Bis-Maleimido Butane) are used; in other embodiments of the present application, heterobifunctional linkers such as alkyne-hydrazide bifunctional linking molecules are used. The type or length of the spacer of the bifunctional linking molecule can be chosen based on the distance of the amino acid residues to be linked and the like.

[0050] The linking molecules in the present application have their most broadest meaning and also encompass disulfide bridging or disulfide re-bridging reagents or disulfide stapling reagents as known in the art, such as the thioester-based bireactive reagents reported in the prior art (Chem. Sci., 2022, 13, 11533-1153); such as the 3Br-5MP reagent (Zhang, Y., Zang, C., An, G. et al. Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylenepyrrolones. Nat Commun 11, 1015 (2020). https: / / doi.org / 10.1038 / s41467-020-14757-4) and the like.

[0051] Structure analysis of CasDinG proteins

[0052] CasDinG is a key component in the CRISPR-Cas acquired immunity type IV-A system of bacteria for resisting foreign invading genetic factors, and the structure thereof is composed of a helicase core and three auxiliary domains, the helicase core includes HD1 (RecA type domain), HD2 (RecA type domain), and the three auxiliary domains are N-terminal domain and two vFe / S, Arch domains inserted in HD1, the HD1 domain contains motif Q, motif I (Walker A), motif Ia, motif II (Walker B), motif III, and the HD2 domain includes motif IV, motif V, and motif VI. The structural information of the corresponding Pea CasDinG (PDB:8e2w) protein can be obtained from the Protein Data Bank (PDB).

[0053] It is found in the present application that removing the N-terminal domain of Pea CasDinG helicase does not affect ATP activity and ssDNA binding activity.

[0054] It is further found in the present application that introducing a covalent linkage between the WalkerA motif, WalkerB motif, motif III and Arch domain in the HD1 domain can reduce the dissociation of the polynucleotide from the helicase, and the Pea CasDinG helicase retains its ability to control the movement of the polynucleotide.

[0055] Example 1: Preparation of Pae CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S mutant combination

[0056] The amino acid sequence of the wild-type Pae CasDinG protein (PDB ID: 8e2w, relevant website: RCSB PDB-8E2W: Structure of CRISPR-Associated DinG) is truncated to obtain SEQ ID NO. 1 (referred to as truncated Pae CasDinG in the present application), the nucleic acid sequence thereof is obtained by in vitro gene synthesis, and the relevant codons are optimized to replace the commonly used codons of E. coli to obtain the nucleic acid sequence of the optimized Pea CasDinG protein, and the sequence is shown as SEQ ID NO. 2. Then, the nucleic acid sequence is inserted into the pET28 expression vector through Nde I and Xho I two restriction enzyme cleavage sites, and after sequence verification, the sequence is correct, finally the recombinant expression plasmid of Pea CasDinG helicase is obtained. Then, the nucleic acid sequences (SEQ ID NO. 4) encoding the mutant combinations E167C, R327C, C144S, C172S, C434S, C448S and C500S are obtained by site-directed mutagenesis through overlap PCR.

[0057] The mutated recombinant plasmid was transformed into BL21(DE3) E. coli expression host by heat shock method. During the process of inducing expression, the host bacteria containing the expression plasmid were first cultured overnight at 37°C with LB medium added with kanamycin, then amplified culture was carried out at 37°C according to the ratio of 1:100, and the culture was stopped when the OD(600) value reached 0.4-0.6 and placed at 4°C for 1 hour of cooling treatment, then 0.5 mM of isopropyl β-D-thiogalactoside (IPTG) was added to induce expression at 16°C for 12-16 hours. Then, the bacteria were collected by centrifugation at 4°C and 15000 rpm, and the bacterial cells were subjected to high-pressure crushing by a high-pressure crusher at 4°C, and then the supernatant was collected by centrifugation at 4°C, and then the target protein was separated and purified step by step through a nickel column, a heparin column, a Q column and a molecular sieve, and finally a large amount of high-purity helicase mutant protein was obtained, and the sequence is as shown in SEQ ID NO. 3.

[0058] 1 μL of 1 M DTT was added to 100 μL of Pea CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S (SEQ ID NO: 3 with mutation combination E167C, R327C, C144S, C172S, C434S, C448S and C500S, stored in 25 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% glycerol, incubated at room temperature for 30 minutes.

[0059] The buffer was replaced by 25 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol buffer through a 0.5 ml Zeba desalting column (7k MWCO) to obtain 100 μl of sample. 1 μL of 10 mM tetramethyladipimidate (TMAD) was added to catalyze the formation of disulfide bond, and the sample was incubated at room temperature for 1 hour with rotation at 20 rpm. A disulfide bond was formed between the cysteines at positions 167 and 327 in the mutated Pea CasDinG sequence. Then, 1 μL of 1 M DTT was added to terminate the reaction to obtain Pea CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S-TMAD. The cross-linking results were analyzed by 4-10% polyacrylamide gel.

[0060] Figure 1 In the figure, band 2 has an upward shift compared to band 1, which represents an intramolecular cross-linking reaction of the mutant protein through the linking molecule under the condition of the same molecular weight. The reaction of the Pea CasDinG mutant with the linking molecule reached a yield of 98%.

[0061] Example 2: Gel shift assay was used to measure the ability of modified Pea CasDinG helicase to bind DNA

[0062] The DNA substrate needed for gel shift assay was prepared by annealing (SEQ ID NO: 5 and SEQ ID NO: 6 were annealed at a molar ratio of 1:1, final concentration of 10 uM), then mixed with wild type Pea CasDinG (PDB ID: 8e2w, relevant website: RCSB PDB-8E2W: Structure of CRISPR-Associated DinG) and Pea CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S (SEQ ID NO: 3 with mutation combination E167C, R327C, C144S, C172S, C434S, C448S and C500S) at a molar ratio of 5:1 in buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol) at room temperature for 0.5 hours, resulting in a final concentration of 2.5 uM Pea CasDinG helicase and 500 nM DNA in the final reaction solution, and the total reaction volume was 20 uL.

[0063] TMAD was added to the corresponding helicase mutant samples to a final concentration of 100 uM, and incubated at room temperature for 1 hour. Subsequently, 4%-10% TBE gel was used for detection, running at 120V for 1.5 hours, and then observing the DNA bands under ultraviolet light by Gel Red staining.

[0064] As shown in Figure 2 Lane 1 represents pure DNA substrate without protein binding, when wild type Pae CasDinG was mixed with DNA substrate at a molar ratio of 5:1, a band of protein binding appeared on the gel, i.e. multiple new bands above lane 1 appeared in lane 2, from bottom to top, representing 1, 2 and 3 proteins binding respectively; while after modification of Pae CasDinG helicase, compared with lane 2, the pure DNA substrate band in lane 3 decreased, and the protein binding band increased, indicating that the modified Pae CasDinG has enhanced binding ability to polynucleotide.

[0065] Example 3: Pea CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S-TMAD has the ability to control the movement of intact DNA construct X through the nanopore

[0066] Preparation as Figure 3The DNA construct X shown:

[0067] First, a primer containing sequences A, C, D, E and F was designed, and then a 1000 base long sequence (G) on the lambda DNA was amplified using the primer, and the obtained PCR product was purified and annealed and hybridized with sequence H at a 1:1.1 molar ratio, thereby obtaining the final DNA construct X.

[0068] A represents 50 T, C represents one iSpC18 spacer, D represents SEQ ID NO: 7, E represents 2 iSpC18 spacers, F represents SEQ IN NO. 8, G represents SEQ ID NO: 9, and H represents SEQ ID NO: 10 with a cholesterol label at the 3' end.

[0069] The prepared DNA construct X (final concentration 0.1 nM) was pre-incubated with Pea CasDinG-E167C, R327C, C144S, C172S, C434S, C448S and C500S (final concentration 10 nM) in a buffer (10 mM Hepes, pH 8.0, 100 Mm KCl, 10% glycerol) at room temperature for 30 minutes, and TMAD was added to the above solution to a final concentration of 100 μM, and incubated at room temperature for 1 hour.

[0070] In a buffer (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0), electrical signal measurements were obtained from Csgg nanopores embedded in DPhPC phospholipid bilayers. After achieving single pore insertion into the phospholipid bilayer, 2 ml of buffer (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0) was flowed through the system to remove residual excess nanopores. Then the pre-incubated sample, ATP (final concentration 2 mM) and MgCl2 (final concentration 10 mM) were flowed into the single nanopore experimental system (total volume 100 μL), and the signal was measured at a constant voltage of +180 mV for 6 h (including a potential 2 s -180 mV voltage reversal).

[0071] Figure 4 showing that the above mutant is capable of controlling the movement of the entire DNA construct X through the nanopore, Figure 5 showing an enlarged view of the part of the DNA movement controlled thereby. Figure 6is the case of simple nucleic acid sample translocation without the control of helicase, such as the arrow marked signal similar to the burr signal represents the simple nucleic acid sample translocation, it can be seen that the speed of simple nucleic acid translocation is very fast (<<1s), and the base information of nucleic acid cannot be identified, but after adding the modified MPK2 helicase, the clear nucleic acid translocation signal can be seen. When using wild type helicase for test, due to its too weak binding force with the nucleic acid sequence to be tested, it slips from the spacer region, so that the signal cannot be measured.

Claims

1. A modified Pae CasDinG helicase, characterized in that, The amino acid sequence of the modified Pae CasDinG helicase is SEQ ID NO: 3; the modified Pae CasDinG helicase is covalently linked by disulfide bond between 167 and 327.

2. A sequencing element or sequencing device comprising the modified Pae CasDinG helicase of claim 1.

3. A sequencing application of the modified Pae CasDinG helicase of claim 1.

4. A method for preparing the modified Pae CasDinG helicase of claim 1, comprising the following steps: 1) providing a Pae CasDinG helicase with an amino acid sequence as set forth in SEQ ID NO: 3; 2) reacting to obtain the modified Pae CasDinG helicase under conditions for forming covalent linkage, the covalent linkage being achieved by disulfide bond between the sulfhydryl groups of cysteines at positions 167 and 327 of the Pae CasDinG helicase.

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