WO2022126304A1

WO2022126304A1 - Modified helicase and application thereof

Info

Publication number: WO2022126304A1
Application number: PCT/CN2020/136031
Authority: WO
Inventors: 王慕旸; 张周刚; 吕伟丽; 王艳双; 陈呈尧
Original assignee: 北京齐碳科技有限公司
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-06-23
Also published as: CN116601297A

Abstract

Provided are a modified F8813 helicase, a construct comprising the modified F8813 helicase, and an application in characterizing a target polynucleotide or controlling the movement of a target polynucleotide passing through pores. Also provided are a method for controlling the movement of a polynucleotide or a method for characterizing the target polynucleotide. The modified F8813 helicase can be used to control the translocation of a DNA chain through transmembrane pores in DNA chain sequencing, and also can realize direct RNA sequencing. Moreover, the modified helicase can remain bound to the polynucleotide for a longer time, thereby reducing the phenomenon of being released from a polynucleotide of a sequenced polynucleotide.

Description

A kind of modified helicase and its application

technical field

The present invention relates to the technical fields of gene sequencing, molecular detection and clinical detection, in particular to a modified helicase, a complex structure comprising the helicase and its function in characterizing target polynucleotides or controlling the penetration of target polynucleotides. Application of the movement of vias.

Background technique

Nanopore sequencing technology refers to a gene sequencing technology that uses a single nucleic acid molecule as a measurement unit and uses nanopores to continuously read its sequence information in real time. The advantage of this sequencing technology is that it is simple to build a library and does not require amplification; the reading speed is fast, and the reading speed of a single molecule can reach tens of thousands of bases per hour; the read length is very long, usually in the thousands of bases; it is possible Direct measurements of RNA and DNA methylation can be performed. These are all beyond the reach of existing next-generation sequencing technologies. However, a common problem with nanopore sequencing is that the translocation of long polynucleotides through the nanopore is so fast that the current levels of individual nucleotides are too short to be resolved. A problem in the sequencing of polynucleotides, especially 500 nucleotides or more, is that the molecular motors that control the movement of polynucleotides may become disengaged from the polynucleotide. This allows the polynucleotide to be pulled through the pore rapidly in an uncontrolled manner in the direction of the applied field.

Nanopore sequencing utilizes nanopores that provide channels for ionic current. Electrophoresis drives the polynucleotide through the nanopore, and as the polynucleotide passes through the nanopore, it reduces the current through the nanopore. A characteristic current is obtained for each passing nucleotide or series of nucleotides, and the recording of the current level corresponds to the polynucleotide sequence. In the "strand sequencing" method, a single polynucleotide strand passes through the pore and enables identification of nucleotides. Strand sequencing can include the use of a nucleotide processing protein, such as a helicase, to control the movement of the polynucleotide through the pore. For example, patent WO2013057495A3 discloses a new method for characterizing target polynucleotides, which includes controlling the movement of target polynucleotides through a pore by Hel308 helicase or molecular motors. Patent US20150065354A1 discloses a method for characterizing target polynucleotides using XPD helicase, the method comprising controlling the movement of target polynucleotides through a pore by the XPD helicase. Patent CN107109380A discloses a modified enzyme, which is a modified Dda helicase that can control the movement of a target polynucleotide through a pore. However, the above-mentioned prior art does not disclose the modified F8813 helicase provided by the present application, which can not only realize the control of its translocation through the transmembrane pore in the DNA strand sequencing, but also realize the direct sequencing of RNA.

The benefits and applications of direct RNA sequencing, such as direct messenger RNA sequencing, provide insight into the dynamics of organisms, including for health screening; for example, the metastatic process of certain cancers and heart disease. Direct RNA sequencing can rapidly identify the type of RNA virus infecting a patient, animal or crop, and has applications in investigating crop disease resistance, determining crop response to stressors such as drought, UV, and salinity, and in embryonic development. Processes of cellular differentiation and decision applications. The problem in direct sequencing of RNAs, especially RNAs of 500 or more nucleotides, is to find suitable molecular motors capable of controlling the translocation of RNAs across transmembrane pores. For characterizing or sequencing polynucleotides, continuous movement of RNA polymers and the ability to read long polymers are required.

Accordingly, the present invention provides a modified helicase that can both control its translocation across a transmembrane pore in DNA strand sequencing and enable direct sequencing of RNA.

SUMMARY OF THE INVENTION

Based on the phenomenon that the translocation of polynucleotides through nanopores in nanopore sequencing is too fast, the sequencing of RNA polynucleotides cannot be performed directly and accurately, and the molecular motors that control the movement of polynucleotides are released from the polynucleotides, the present invention provides A modified helicase that can both control the translocation speed of DNA strands through a transmembrane pore and improve the accuracy of direct RNA sequencing, and the modified helicase The helicase can remain bound to the polynucleotide for a longer time, reducing the phenomenon of unwinding from the polynucleotide being sequenced.

The present inventors found that the reduction or opening and closing of the opening size of the polynucleotide binding domain on the F8813 helicase does not prevent its binding to the polynucleotide. Accordingly, the present invention introduces cysteine residues and/or at least one unnatural amino acid in the F8813 helicase to allow the linkage of two or more moieties to reduce the size of the opening or close the opening. Once the F8813 helicase modified according to the present invention is bound to a polynucleotide, it is able to control the speed of movement of the target polynucleotide without the need for unwinding or dissociation. Specifically, the F8813 helicase modified according to the present invention will bind strongly to long polynucleotides (eg, polynucleotides comprising 500 or more nucleotides), and will control the speed of movement of the target polynucleotide, Especially during strand sequencing. The specific plans are as follows:

The first aspect of the present invention provides a modified F8813 helicase, the F8813 helicase comprises a polynucleotide binding domain, and the F8813 helicase comprises a variant of SEQ ID NO: 1 , the variant of SEQ ID NO: 1 is included in the 2A domain (preferably at position E250 to H310), the Ratchet domain (preferably at position L580 to G600) and/or the HLH domain (preferably at position L580 to G600) of SEQ ID NO: 1 At least one or more (

eg

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) cysteine residues and/or at least one or more (

eg

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) unnatural amino acids to reduce the opening size of the polynucleotide binding domain, wherein the F8813 helicase retains its ability to control movement of polynucleotides.

Preferably, at least one cysteine residue and/or at least one unnatural amino acid is introduced in any of the following groups:

(a) 2A domain;

(b) Ratchet domain;

(c) HLH domain;

(d) 2A domain and Ratchet domain;

(e) 2A domain and HLH domain;

(f) Ratchet domain and HLH domain;

(g) 2A domain, Ratchet domain and HLH domain.

Preferably, at least one or more (

eg

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) cysteine residues are introduced in SEQ ID NO: 1 , or introduce at least one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) unnatural amino acids, or introduce at least one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) cysteine residues and unnatural amino acids.

Preferably, S272, A273, E274, E281, D284, E285, L287, E288, N289, S290, E291, D293, T294, A300, R303, D284, T314, T315, P316, L317 of SEQ ID NO: 1 are included , R318, L320, E322, R326, G328, S589, S589, K691, D694, R695 or K698 in any one or two or more positions to introduce at least one cysteine residue and/or at least one non-natural amino acid.

Further preferably, the variant of SEQ ID NO: 1 comprises introducing at least one cysteine residue and/or at least one unnatural amino acid at the D284 and/or S589 position of SEQ ID NO: 1.

More preferably, the variant of SEQ ID NO: 1 also includes S272, A273, E274, E281, D284, E285, L287, E288, N289, S290, E291, D293, T294, At least one cysteine is introduced at any one or two or more positions of A300, R303, T314, T315, P316, L317, R318, L320, E322, R326, G328, S589, K691, D694, R695 or K698 residues and/or at least one unnatural amino acid.

In order to improve the stability of the binding of the F8813 helicase of the present invention to the target polynucleotide, and reduce the ability to be freed from the target polynucleotide: the introduced cysteine and the cysteine are connected to each other, and the introduced cysteine Interconnection between unnatural amino acids and unnatural amino acids, between introduced cysteine and unnatural amino acids, between introduced cysteine and natural amino acids, or between introduced unnatural amino acids and natural amino acids connected to each other.

Preferably, any number and combination of two or more introduced cysteines and unnatural amino acids can be interconnected. For example, 3, 4, 5, 6, 7, 8 or more cysteines and/or unnatural amino acids can be linked to each other. One or more cysteines can be linked to one or more cysteines. One or more cysteines can be linked to one or more unnatural amino acids such as Faz. One or more unnatural amino acids such as Faz can be linked to one or more unnatural amino acids such as Faz. One or more cysteines can be attached to one or more natural amino acids on the helicase. One or more unnatural amino acids such as Faz can be linked to one or more natural amino acids on the helicase.

Preferably, the connection can be any connection mode, including a temporary connection or a permanent connection.

In one embodiment of the invention, the linkage may be temporary, eg, non-covalent linkage. Of course, even a brief ligation is sufficient to reduce the release of the target polynucleotide from the F8813 helicase.

In another embodiment of the present invention, the linkage may be permanent, such as a covalent linkage. Covalent attachment can be carried out using chemical crosslinkers, which can vary in length from one carbon (phosgene type linker) to multiple Angstroms. For example maleimides, active esters, succinimides, azides, alkynes such as dibenzocyclooctynol (DIBO or DBCO), difluorocycloalkynes and linear alkynes, and the like. Another example is polyethylene glycol (PEGs), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons or polyamides Linear molecules such as TMAD, etc., and catalytic reagents such as TMAD, can be linked by -S-S bonds.

In a specific embodiment of the present invention, bismaleimides PEG3 and/or PEG4 are used to covalently link the introduced cysteine residues to the cysteine residues.

Further preferably, the variant of SEQ ID NO: 1 further comprises that at least one or more cysteines of SEQ ID NO: 1 are substituted. In one embodiment of the invention, cysteine is replaced with alanine, serine or valine.

Preferably, the substituted one or more cysteines are one or a combination of two or more of C172, C217, C246, C256, C301, C469, C527 or C594.

Preferably, the F8813 helicase comprises removing the C-terminal HLH domain of the F8813 helicase. Further preferred, the amino acid sequence from A644 to Y729 of the C-terminal HLH domain is removed.

The unnatural amino acids of the present invention include but are not limited to 4-azido-L-phenylalanine (Faz), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine Acid, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenyl)-L-alanine, O-2-propyne-1 -yl-L-tyrosine, 4(dihydroxyboronyl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2- Amino-3-{4-[(propan-2-ylsulfanyl)carbonyl]phenyl}propionic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanyl Propionyl)amino]phenyl}propionic acid, O-methyl-L-tyrosine, 4-amino-L-phenylalanine, 4-cyano-L-phenylalanine, 3-cyano- L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(trifluoromethyl)tyrosine Acid, 4-Nitro-L-Phenylalanine, 3-Hydroxy-L-Tyrosine, 3-Amino-L-Tyrosine, 3-Iodo-L-Tyrosine, 4-Isopropyl-L -Phenylalanine, 3-(2-naphthyl)-L-alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthalen-2-ylamino ) propionic acid, 6-(methylsulfanyl) norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-hydroxy-3-(4-hydroxyphenyl) ) propionic acid, (2R)-2aminocaprylate 3-(2,2'-dipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinoline yl)propionic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl) yl)sulfanyl]propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-dimethoxy-2-nitro Benzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, O-(2-nitrobenzyl)- L-tyrosine, 2-nitrophenylalanine, 4-[(E)-phenyldiazenyl]-L-phenylalanine, 4-[3-(trifluoromethyl)-3H -Diaziridinyl-3yl]-D-phenylalanine, 2-amino-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]propionic acid, (2S) -2-Amino-4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanoic acid, (2S)-3-[(6-acetylnaphthalen-2-yl)amino]-2- Aminopropionic acid, 4(carboxymethyl)phenylalanine, 3-nitro-L-tyrosine, O-thio-L-tyrosine, (2R)-6-acetamido-2-aminohexyl Acetate, 1-Methylhistidine, 2-Aminononanoic acid, 2-Aminodecanoic acid, L-Homocysteine, 5-Sulfanylnorvaline, 6-Sulfanyl-L- Norleucine, 5-(methylsulfanyl)-L-norvaline, N6-{[(2R,3R )-3-methyl-3,4-dihydro-2H-pyrrolidin 2-yl]carbonyl}-L-lysine, N6-[(benzyloxy)carbonyl]lysine, (2S)-2 -Amino-6-[(cyclopentylcarbonyl)amino]hexanoic acid, N6-[(cyclopentyloxy)carbonyl]-L-lysine, (2S)-2-amino-6-{[(2R )-tetrahydrofuran-2-ylcarbonyl]amino}hexanoic acid, (2S)-2-amino-8-[(2R,3S)-3-ethynyltetrahydrofuran-2-yl]-8-oxyoctanoic acid, N6- (tert-Butoxycarbonyl)-L-lysine, (2S)-2-hydroxy-6-({[(2-methyl-2-propanyl)oxy]carbonyl}amino)hexanoic acid, N6- [(allyloxy)carbonyl]lysine, (2S)-2-amino-6-({[(2-azidobenzyl)oxy]carbonyl}amino)hexanoic acid, N6L-prolyl- L-Lysine, (2S)-2-amino-6-{[(prop-2-yn-1-yloxy)carbonyl]amino}hexanoic acid or N6-[(2azidoethoxy)carbonyl ]-L-Lysine.

Preferably, the amino acid sequence of the F8813 helicase is the amino acid sequence of SEQ ID NO: 1 or its variant or the amino acid sequence of SEQ ID NO: 1 or its variant with at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 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%, or at least 99.9% homology, and have the ability to control polynucleotide movement.

Preferably, the F8813 helicase is further modified to reduce the negative charge on its surface.

Preferably, the F8813 helicase further comprises substitutions that increase the net positive charge. Preferably, the F8813 helicase further comprises substitution or modification of surface negatively charged amino acids, polar or non-polar amino acids. Further preferably, the substitution includes the substitution of positively charged amino acids, uncharged amino acids for negatively charged amino acids, uncharged amino acids, aromatic amino acids, polar or non-polar amino acids.

Wherein, described positively charged amino acid, uncharged amino acid, polar, non-polar amino acid or aromatic amino acid can be natural or non-natural amino acid, it can be synthetic or modified natural amino acid .

Preferably, the F8813 helicase also comprises:

(a) at least one amino acid that interacts with one or more nucleotides in single-stranded DNA, RNA or double-stranded DNA, RNA is substituted; and/or,

(b) at least one amino acid that interacts with the transmembrane pore is substituted;

The F8813 helicase has the ability to control the movement of polynucleotides.

Further preferably, at least one amino acid that interacts with the sugar and/or base of one or more nucleotides in single-stranded DNA, RNA or double-stranded DNA, RNA is replaced with an amino acid comprising a larger side chain.

The larger side chains include an increased number of carbon atoms, have an increased length, have an increased molecular volume, and/or have an increased van der Waals volume. The larger side chain increases (i) electrostatic interactions between the at least one amino acid and one or more nucleotides in the single- or double-stranded DNA; (ii) hydrogen bonding and/or (iii) ) cation-pi interaction. The amino acid of the larger side chain is not alanine (A), cysteine (C), glycine (G), selenocysteine (U), methionine (M), aspartate acid (D) or glutamic acid (E).

Further preferably, at least one amino acid that interacts with the phosphate group of one or more nucleotides in single-stranded DNA, RNA or double-stranded DNA, RNA is substituted.

The second aspect of the present invention provides a construct comprising at least one or more of the F8813 helicases of the present invention.

Preferably, the construct further comprises a polynucleotide binding moiety.

Preferably, the F8813 helicase is linked to the polynucleotide binding moiety, and the construct has the ability to control the movement of the polynucleotide.

Preferably, the polynucleotide binding moiety may be a moiety that binds to the base of the polynucleotide, and/or a moiety that binds to the sugar of the polynucleotide, and/or a moiety that binds to the phosphate of the polynucleotide.

Preferably, the F8813 helicase and polynucleotide binding moieties that make up the construct can be prepared separately and then directly linked. The construct can also be directly prepared by genetic fusion, for example, by linking the nucleotides encoding the F8813 helicase to the polynucleotide binding portion, and then transferring it into a host cell for expression and purification.

Further preferably, the polynucleotide binding moiety is a polypeptide that can bind to polynucleotides, including but not limited to eukaryotic single-stranded binding proteins, bacterial single-stranded binding proteins, archaeal single-stranded binding proteins, and viral single-stranded binding proteins. One or a combination of two or more binding proteins or double-stranded binding proteins.

In a specific embodiment of the present invention, the described polynucleotide binding part includes but is not limited to any one shown in Table 1:

Table 1 Binding moieties that bind to polynucleotides

The third aspect of the present invention provides a nucleic acid encoding the F8813 helicase or the construct of the present invention.

The fourth aspect of the present invention provides an expression vector, the expression vector comprising the nucleic acid of the present invention.

Preferably, the nucleic acid is operably linked to a regulatory component in an expression vector, wherein the regulatory component is preferably a promoter.

In a specific embodiment of the present invention, the promoter is selected from T7, trc, lac, ara or λ _L .

Preferably, the expression vector includes but is not limited to plasmid, virus or phage.

The fifth aspect of the present invention provides a host cell, the host cell comprising the nucleic acid of the present invention or the expression vector of the present invention.

Preferably, the host cells include but are not limited to Escherichia coli.

In a specific embodiment of the present invention, the host cell is selected from BL21(DE3), JM109(DE3), B834(DE3), TUNER, C41(DE3), Rosetta2(DE3), Origami, Origami B, etc. .

The sixth aspect of the present invention provides a preparation method of the F8813 helicase of the present invention, comprising providing SEQ ID NO: 1, and introducing at least one cysteine residue and/or into SEQ ID NO: 1 At least one unnatural amino acid obtains a variant of SEQ ID NO: 1 to reduce the opening size of the polynucleotide binding domain of the F8813 helicase, wherein the F8813 helicase retains its ability to control polynucleotide movement.

The seventh aspect of the present invention provides a method for preparing the F8813 helicase of the present invention, comprising culturing the host cell of the present invention, inducing expression, and purifying to obtain the F8813 helicase.

In a specific embodiment of the present invention, the method includes obtaining a nucleic acid sequence encoding the F8813 helicase according to the amino acid sequence of the F8813 helicase of the present invention, cleaving it into an expression vector and transforming it into E. coli , induced expression and purification to obtain F8813 helicase.

The eighth aspect of the present invention provides a method for regulating the opening size of a polynucleotide binding domain of a helicase, the method comprising adding the F8813 helicase of the present invention or the construct of the present invention contact with a polynucleotide. It is preferred to reduce the size of the polynucleotide binding domain opening.

The ninth aspect of the present invention provides a method for controlling the movement of a polynucleotide, the method comprising contacting the F8813 helicase of the present invention or the construct of the present invention with a polynucleotide.

Preferably, the control of the movement of the polynucleotide is the movement of the control polynucleotide through the pore. The pores are nanopores, and the nanopores are transmembrane pores. The pores may be natural or artificial, including but not limited to protein pores, polynucleotide pores, or solid state pores.

In a specific embodiment of the present invention, the transmembrane pore is selected from biological pore, solid state pore or pore hybridized between biological and solid state.

In a specific embodiment of the present invention, the pores include but are not limited to those derived from M. smegmatis porin A, M. smegmatis porin B, M. smegmatis porin C, smegmatis porin Mycobacterial porin D, hemolysin, lysin, interleukin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, WZA or Neisseria autotransport lipoprotein and the like.

Preferably, the method may comprise one or more F8813 helicases jointly controlling the movement of the polynucleotide.

A tenth aspect of the present invention provides a method for characterizing a target polynucleotide, the method comprising:

1) contacting the F8813 helicase of the present invention or the construct of the present invention with the target polynucleotide and the pore, so that the F8813 helicase or the construct controls the movement of the target polynucleotide through the pore;

and II) obtaining one or more characteristics of the interaction of nucleotides in the target polynucleotide with the pore to characterize the target polynucleotide.

Preferably, steps I) and II) are repeated one or more times.

Preferably, any number of the F8813 helicases of the present invention can be used in the method. Preferably, there may be one or more, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. Wherein, the two or more F8813 helicases of the present invention can be the same or different. Wild-type F8813 helicases or other types of helicases may also be included. Further, two or more helicases can be linked or just arranged to play the function of controlling the movement of the polynucleotide by binding to the polynucleotide respectively.

Preferably, the method further comprises the step of applying a potential difference across the pore in contact with the helicase or construct, and the polynucleotide of interest.

Preferably, the pores are structures that allow hydrated ions to flow from one side of the membrane to the other layer of the membrane, driven by an applied electrical potential. Further preferably, the pores are nanopores, and the nanopores are transmembrane pores. The transmembrane pore provides a channel for the movement of the target polynucleotide. Further preferably, the pores are selected from biological pores, solid-state pores or hybrid pores of biological and solid-state.

Said membrane may be any membrane existing in the prior art, preferably an amphiphilic layer, ie a layer formed of amphiphilic molecules such as phospholipids having at least one hydrophilic part and at least one lipophilic or hydrophobic part , amphiphilic molecules can be synthetic or naturally occurring. Further preferably, the membrane is a lipid bilayer membrane. The target polynucleotide can be attached to the membrane using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer, the polynucleotide is preferably attached to the membrane via a polypeptide present in the membrane or via a hydrophobic anchor present in the membrane. Among them, the hydrophobic anchor is preferably lipid, fatty acid, sterol, carbon nanotube or amino acid.

Preferably, when a force (eg, a voltage) is applied to the pore, the rate of passage of the target polynucleotide through the pore is controlled by the F8813 helicase or construct, resulting in an identifiable stable current level for target determination Characterization of polynucleotides.

Preferably, the target polynucleotide is single-stranded, double-stranded or at least partially double-stranded.

Further preferably, the target polynucleotide can be modified by means of tags, spacers, methylation, oxidation or damage.

In a specific embodiment of the present invention, the target polynucleotide is at least partially double-stranded. wherein the double-stranded portion constitutes a Y-adapter structure comprising a leader sequence that preferentially screws into the pore.

Further preferably, the length of the target polynucleotide may be 10-100,000 or more.

In a specific embodiment of the present invention, the length of the target polynucleotide can be at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 50,000, or at least 100,000, etc.

Preferably, the helicase is incorporated into the internal nucleotides of the single-stranded polynucleotide.

Preferably, the target polynucleotide is DNA or RNA. That is, DNA, RNA, DNA-RNA junction sequences, DNA-RNA hybrid sequences, etc. composed of ribonucleotides and/or deoxyribonucleotides as units.

Preferably, when the target polynucleotide is RNA, in order to improve the ability and efficiency of the RNA to be sequenced passing through the pore, the RNA is modified to include a non-RNA polynucleotide.

Preferably, the step of RNA modification comprises linking the DNA leader region to the 3' end of the RNA to be tested. It also includes the step of reverse transcription of the RNA to be tested

Preferably, the one or more features are selected from the source, length, identity, sequence, secondary structure of the target polynucleotide, or whether the target polynucleotide is modified. Further preferably, the one or more features are performed by electrical and/or optical measurements.

Further preferably, electrical and/or optical signals are generated by electrical and/or optical measurements, and each nucleotide corresponds to a signal level, and the electrical and/or optical signals are then converted into nucleotide characteristics.

In a specific embodiment of the present invention, the electrical measurement includes, but is not limited to, current measurement, impedance measurement, tunnel measurement, wind tunnel measurement, or field effect transistor (FET) measurement, and the like.

The electrical signal according to the present invention is selected from measurements of current, voltage, tunneling, resistance, potential, conductivity or lateral electrical measurements.

In a specific embodiment of the present invention, the electrical signal is a current passing through the hole.

Preferably, the characterization further includes applying an improved Viterbi algorithm.

An eleventh aspect of the present invention provides a product for characterizing a target polynucleotide, the product comprising one or more of the F8813 helicases of the present invention, one or more of the constructs of the present invention , one or more nucleic acids of the present invention, one or more expression vectors of the present invention, or one or more host cells of the present invention, and one or more pores.

Preferably, the pore forms a complex with the F8813 helicase or construct.

In a specific embodiment of the invention, the product comprises a plurality of F8813 helicases or a plurality of constructs, and a plurality of wells.

Preferably, the product is selected from kits, devices or sensors.

Further preferably, the kit also includes a chip comprising lipid bilayers. The pores span the lipid bilayer.

The kits of the present invention comprise one or more lipid bilayers, each lipid bilayer comprising one or more of said pores.

The kits of the present invention also include reagents or devices for carrying out the characterization of the polynucleotide of interest. Preferably, the reagents include buffers and tools required for PCR amplification.

The twelfth aspect of the present invention provides the F8813 helicase, the construct, the nucleic acid, the expression vector, the host cell or the product described in the present invention in characterizing the target Applications in polynucleotides or controlling the movement of a polynucleotide of interest through a pore.

The thirteenth aspect of the present invention provides a kit for characterizing a target polynucleotide, the kit comprising the F8813 helicase of the present invention, the construct, the nucleic acid, the The expression vector or the host cell, and well.

The fourteenth aspect of the present invention provides a device for characterizing a target polynucleotide, the device comprising the F8813 helicase of the present invention, the construct, the nucleic acid, the expression The vector or the host cell, and the pores.

Preferably, the device includes a sensor that supports the plurality of pores and transmits a signal that the pores interact with the polynucleotide, and at least one memory for storing the polynucleotide of interest, and necessary for carrying out the characterization process. solution.

Preferably, the device comprises multiple F8813 helicases and/or multiple constructs, and multiple wells.

A fifteenth aspect of the present invention provides a sensor for characterizing a target polynucleotide, the sensor comprising forming a complex between the pore and the F8813 helicase or the construct of the present invention.

Preferably, the pore and the helicase or construct are contacted in the presence of the polynucleotide of interest and an electrical potential is applied across the pore. Said potential is selected from voltage potential or chemical potential.

A sixteenth aspect of the present invention provides a method for forming a sensor for characterizing a target polynucleotide, comprising forming a complex between the pore and the F8813 helicase or the construct of the present invention, thereby A sensor is formed that characterizes the polynucleotide of interest.

The seventeenth aspect of the present invention provides two or more helicases linked to a polynucleotide, wherein at least one of the two or more helicases is the F8813 of the present invention helicase.

The eighteenth aspect of the present invention provides an F8813 helicase oligomer, wherein the F8813 helicase oligomer comprises one or more of the F8813 helicases of the present invention.

Preferably, the F8813 helicase oligomer may further comprise wild-type F8813 helicase or other types of helicases. Wherein, the other types of helicases can be Hel308 helicase, XPD helicase, Dda helicase, TraI helicase or TrwC helicase and the like.

Preferably, between the F8813 helicase and the wild-type F8813 helicase, between the F8813 helicase and the F8813 helicase, between the wild-type F8813 helicase and the wild-type F8813 helicase, and between the F8813 helicase With other types of helicases or between wild-type F8813 helicases and other types of helicases, they can be connected or arranged in a head-to-head, tail-to-tail or head-to-tail manner.

Preferably, the F8813 helicase oligomer comprises two or more F8813 helicases of the present invention, wherein the F8813 helicases may be different or the same.

The "F8813 helicase", "construct" or "pore" of the invention can be modified to aid in identification or purification, for example by adding histidine residues (His tag), aspartic acid residues base (asp tag), streptavidin tag, Flag tag, SUMO tag, GST tag or MBP tag, or by adding a signal sequence to facilitate their secretion from cells in which the polypeptide does not naturally contain the signal sequence . An alternative way of introducing a genetic tag is to chemically link the tag to a natural or artificial site on the F8813 helicase, pore or construct.

The "nucleotides" in the present invention include but are not limited to: adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytosine Nucleoside monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), and deoxycytidine monophosphate (dCMP). Preferably, the nucleotides are selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.

The term "two or more" in the present invention includes two, three, four, five, six, seven, eight or more and the like.

The "plurality" in the present invention includes, but is not limited to, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, and so on.

In the present invention, "at least one" includes, but is not limited to, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, and so on.

As used herein, "and/or" includes any number of the listed items and combinations of any number.

In the present invention, "comprising" or "comprising" is an open-ended description containing the specified elements or steps described, as well as other specified elements or steps that are not substantially affected. When used in this application to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but Still have the activity described in the present invention (eg, its ability to control movement of polynucleotides, etc.).

The "opening" in the present invention refers to the opening of the polynucleotide binding domain of the wild-type F8813 helicase itself, and may also refer to the opening of the polynucleotide binding portion bound to the F8813 helicase. An opening for dissociation of the polynucleotide from the F8813 helicase, and the opening may not always be present, but at least in one conformational state comprises at least one opening. The "modified F8813 helicase" of the present invention or a construct comprising the modified F8813 helicase comprises one or more regions that bind to polynucleotides, including one or more polynucleotides that it carries An acid binding domain or one or more polynucleotide binding moieties. The "modified F8813 helicase" or a construct comprising the modified F8813 helicase contains one or more openings. The F8813 helicase is modified so that two or more moieties are attached on the same monomer of the helicase to reduce the size of the opening.

The F8813 helicase of the present invention is derived from Methanosarcinathermophila.

The modified F8813 helicases described in the present invention are modified compared to the corresponding wild-type helicases or native helicases. The helicases of the present invention are artificial or non-natural.

The F8813 helicase of the present invention is a useful tool for controlling the movement of target polynucleotides during strand sequencing, and when provided with the usual necessary components to facilitate movement, the F8813 helicase follows DNA or RNA with 3 ' to the 5' end, but the orientation of the DNA or RNA in the pore (depending on which end of the DNA or RNA is captured) means that the F8813 helicase can be used against the direction of the applied field or along the The direction of the field moves the DNA or RNA into the well. Furthermore, the polynucleotide binding domain or polynucleotide binding moiety on the F8813 helicase or construct can be effectively reduced by introducing cysteine residues and/or at least one unnatural amino acid in the wild-type F8813 helicase The size or opening of the opening, as well as the size or opening of the opening in which the target polynucleotide is unwound, significantly reduces the ability of the F8813 helicase to dissociate from the target polynucleotide and improves control of the passage of the target polynucleotide through the pore. ability.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1: shows the SDS-PAGE gel electrophoresis image of purified F8813_C172V/C217A/D284C/S589C/C594A. Among them, M is Marker (KDa), and lane 1 is the electrophoresis result of F8813_C172V/C217A/D284C/S589C/C594A helicase.

Figure 2: Diagram of DNA construct A used in the examples, wherein SEQ ID NO: 3 (labeled A) has its 5' end joined to four iSpC3 spacers (labeled B), the spacers (labeled B) ) is ligated to the 3' end of SEQ ID NO: 4 (labeled C), the 5' end of the SEQ ID NO: 4 (labeled C) is ligated to SEQ ID NO: 5 (labeled D), the construct's The region of SEQ ID NO:6 (labeled E) hybridizes to SEQ ID NO:7 (labeled F, which has a 3' cholesterol tether).

Figure 3: shows the F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 reaction mixture (SEQ ID NO: 1, mutations C172V/C217A/D284C/S589C/C594A linked by bismaleimide PEG3 linker) and F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG4 reaction mixture (SEQ ID NO: 1, mutation C172V/C217A/D284C/S589C/C594A via bismaleimide PEG4 linker ligated) Coomassie-stained 4-20% SDS-PAGE gel. Lane M shows an appropriate protein ladder (mass unit markers are shown to the right of the gel). Lane 1 contained 5 μL of F8813_C172V/C217A/D284C/S589C/C594A monomer (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A) at about 6 μM. Lane 2 contained 5 μL of approximately 6 μM F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (SEQ ID NO: 1, mutant C172V/C217A/D284C/S589C/C594A via bismaleimide PEG3 connector connection). Lane 3 contained 5 μL of approximately 6 μM F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG4 (SEQ ID NO: 1, mutant C172V/C217A/D284C/S589C/C594A via bismaleimide PEG4 connector connection). It is clear from the gel that the reaction for bismaleimide PEG3 or PEG4 linker attachment reaches almost 100% yield. Arrow 1 corresponds to F8813_C172V/C217A/D284C/S589C/C594A monomer (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A), arrow 2 corresponds to F8813_C172V/C217A/D284C/S589C/C594A-double Maleimide PEG3 (SEQ ID NO: 1 with mutations C172V/C217A/D284C/S589C/C594A linked to bismaleimide-PEG3). Arrow 3 corresponds to F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide-PEG4 (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A linked to bismaleimide-PEG4) .

Figure 4: Shows when the helicase (F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (SEQ ID NO: 1, mutation C172V/C217A/D284C/S589C/C594A passes through the bismaleimide PEG3 (SEQ ID NO: 1) Amine PEG3 linker ligation)) Example current traces (y-axis coordinate = current(pA, 0 to 240), x-axis coordinate = time(h:m:s, 14h:01m:09.3s to 14h:01m:33.3s)).

Figure 5: Shows an enlarged view of the region of helicase-controlled DNA movement shown in the current trace graph of Figure 4 (y-axis coordinate = current (pA, 0 to 100), x-axis coordinate = time (h:m) :s, 14h:01m:12.4s to 14h:01m:21.9s).

Figure 6: Schematic diagram of fluorescence assay to detect F8813 helicase enzymatic activity. Among them, the fluorogenic substrate strand has a 3' end ssDNA overhang, and a 50-base hybridized dsDNA portion. a), including the upper part of the main chain (B) with carboxyfluorescein (C) at the 5' end, and the hybridized complementary strand (D) with a black hole quencher (BHQ-1) base at the 3' end (E). Also included was a 0.5 [mu]M capture strand (F) complementary to the shorter strand (D). Helicase (100 nM) added to the substrate in the presence of ATP (5 mM) and MgCl (5 mM) as shown in b) and c) (A) is attached to the 3' end of the fluorogenic substrate In part, after moving along the backbone and unwinding the complementary strand, the excess capture strand preferentially anneals to the complementary strand DNA to prevent reannealing of the original substrate with lost fluorescence. As shown in d), after adding an excess of the capture strand (G) that is completely complementary to the main chain, part of the dsDNA that has not been unwound has a chain unwinding effect due to the presence of excess G, and finally all dsDNA is unwound, and the fluorescence value reaches Highest.

Fig. 7 is a graph showing the time-dependent displacement ratio of dsDNA in a buffer containing 400 mM NaCl.

Figure 8: Diagram of DNA-RNA construct B used in the Examples, wherein SEQ ID NO: 13 (labeled G) has its 3' end linked to 20 iSpC3 spacers (labeled A) its 5' end is linked to 4 iSpC3 spacers (labeled B) linked to the 3' end of SEQ ID NO: 14 (labeled C) and 5' to SEQ ID NO: 14 (labeled C) End-ligated to the RNA single strand SEQ ID NO: 15 (labeled D), the SEQ ID NO: 16 (labeled E) region of the construct was linked to SEQ ID NO: 7 (labeled F) with a 3' cholesterol tether ) hybrid.

Figure 9: When the F8813 helicase (F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (SEQ ID NO: 1 with the mutations C172V/C217A/D284C/S589C/C594A) and bismaleimide Amine-PEG3 linkage)) Example current traces (y-axis coordinate = current(pA, -10 to 200), x-axis coordinate when translocation of DNA-RNA construct B through nanopore 8MspA (SEQ ID NO: 8) is controlled) = time(h:m:s, 16h:32m:51.6s to 16h:33m:03.6s).

Figure 10: Enlarged view of the region of F8813 helicase-controlled RNA movement shown in the current trace plot of Figure 9 (y-axis coordinate = current (pA, -10 to 200), x-axis coordinate = time (h:m:s) , 16h:33m:01.3s to 16h:33m:03.3s).

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The equipment and reagents used in each example are conventionally commercially available.

Example 1 Preparation of F8813 helicase

1. Sequence acquisition

Cysteines were introduced at positions 284 and 589 of SEQ ID NO: 1 and substituted at positions 172, 217 and 594 to obtain a variant of SEQ ID NO: 1, namely C172V/C217A SEQ ID NO: 1 of /D284C/S589C/C594A.

The coding sequence of SEQ ID NO: 1 of C172V/C217A/D284C/S589C/C594A was obtained and optimized to obtain SEQ ID NO:2.

2. Materials and methods

The recombinant plasmid containing the F8813 helicase sequence (SEQ ID NO: 2) was transformed into BL21 (DE3) competent cells by heat shock, and the recovered bacterial solution was coated on an ampicillin-resistant solid LB plate and cultured at 37°C overnight. Monoclonal colonies were inoculated into 100 ml of liquid LB medium containing ampicillin resistance and cultured at 37°C. 1% of the inoculum was transferred to ampicillin-resistant LB liquid medium for expansion culture, and cultured at 37° C. and 200 rpm, and the OD ₆₀₀ value was continuously measured. When OD ₆₀₀ =0.6-0.8, the culture medium in LB medium was cooled to 18°C, and isopropyl thiogalactoside (IPTG) was added to induce expression, so that the final concentration reached 1 mM. After 12-16 h, the bacteria were collected at 18°C. Bacteria were disrupted by high pressure, purified by FPLC method, and samples were collected.

3. Results

Figure 1 shows an SDS-PAGE gel electropherogram of purified F8813 helicase (variant of SEQ ID NO: 1).

Example 2 The ability of F8813 helicase to unwind hybrid dsDNA was demonstrated using fluorescence assay to detect enzymatic activity

1. Materials and methods

As shown in a) of Figure 6, the fluorogenic substrate strand (final concentration 100 nM) has a 3'-end ssDNA overhang, and a 50-base hybridized dsDNA portion. The upper part of the main chain has carboxyfluorescein (5'FAM-SEQ ID NO: 9) at the 5' end, and the hybridized complementary strand has a black hole quencher (BHQ-1) base (SEQ ID NO: 9) at the 3' end. NO:10--BHQ-3'). When the hybridized fluorescence from fluorescein is quenched by localized BHQ-1, the substrate is essentially non-fluorescent. Also included was a 0.5 μM capture strand complementary to the shorter strand (SEQ ID NO: 11). As shown in b) and c), F8813 helicase (100 nM) added to the substrate in the presence of ATP (5 mM) and MgCl ₂ (5 mM) was attached to the 3' end portion of the fluorogenic substrate along the After the backbone has moved and the complementary strand has been unwound, the excess capture strand preferentially anneals to the complementary strand DNA to prevent reannealing of the original substrate with lost fluorescence. At the same time, there is still a certain amount of hybrid dsDNA in the system that has not been unwound by F8813 helicase. As shown in d), after adding an excess of the capture strand G (SEQ ID NO: 12) that is completely complementary to the main chain, part of the dsDNA that has not been unwound has a chain unwinding effect due to the presence of excess G, and finally all dsDNA is unwound spin, the fluorescence value reaches the highest value.

2. Results

Figure 7 shows a graph of time-dependent dsDNA unwinding ratio changes in buffer containing 400 mM NaCl (10 mM Hepes pH 8.0, 5 mM ATP, 5 mM _MgCl2 , 100 nM fluorogenic substrate DNA, 0.5 [mu]M capture DNA). NC-Buffer is a negative control without F8813 helicase added.

Example 3

This example describes the synthesis of F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 helicase (SEQ ID NO: 1 with the mutations C172V/C217A/D284C/S589C/C594A and bismaleimide -PEG3 linkage) and F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG4 helicase (SEQ ID NO: 1 with bismaleimide with mutation C172V/C217A/D284C/S589C/C594A -PEG4 linking) method. In this case, a covalent linkage was formed between cysteines 284 and 589 in the primary sequence of F8813 by reaction with a bismaleimide PEG3 or bismaleimide PEG4 linker.

1. Materials and methods

5 μl of 1M DTT was added to 1 ml of F8813 (C172V/C217A/D284C/S589C/C594A) (SEQ ID NO: 1, mutant C172V/C217A/D284C/S589C/C594A, stored in 50 mM Tris HCl pH 8.0, 100 mM NaCl, 50% glycerol , 1 mM DTT, 1 mM EDTA) and spun at 20 rpm for 30 min at room temperature. A 1.25 mL sample was obtained by buffer exchange to PBS buffer (pH 7.2) through a PD-10 desalting column. After addition of 20 μL of bismaleimide PEG3 or bismaleimide PEG4, the mixture was incubated at room temperature for 60 minutes with rotation at 20 rpm. To stop the reaction, add 5 μl of 1 M DTT to quench any remaining maleimide. Results were analyzed using 4-20% polyacrylamide gels.

2. Results

Figure 3 shows the F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 reaction mixture (SEQ ID NO: 1, mutation C172V/C217A/D284C/S589C/C594A via a bismaleimide PEG3 linker ligation) and F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG4 reaction mixture (SEQ ID NO: 1, mutation C172V/C217A/D284C/S589C/C594A ligated via a bismaleimide PEG4 linker ) of a Coomassie-stained 4-20% SDS-PAGE gel. Lane M shows an appropriate protein ladder (mass unit markers are shown to the right of the gel). Lane 1 contains 5 μL of F8813_C172V/C217A/D284C/S589C/C594A monomer (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A) at about 6 μM. Lane 2 contained 5 μL of approximately 6 μM F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (SEQ ID NO: 1, mutant C172V/C217A/D284C/S589C/C594A via bismaleimide PEG3 connector connection). Lane 3 contained 5 μL of approximately 6 μM F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG4 (SEQ ID NO: 1, mutant C172V/C217A/D284C/S589C/C594A via bismaleimide PEG4 connector connection). It is clear from the gel that the reaction to attach the bismaleimide PEG3 or PEG4 linker achieves almost 100% yield. Then F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide-PEG3 (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A was linked to bismaleimide-PEG3) and F8813_C172V /C217A/D284C/S589C/C594A-bismaleimide-PEG4 (SEQ ID NO: 1 with mutation C172V/C217A/D284C/S589C/C594A linked to bismaleimide-PEG4) buffer exchanged to 20 mM HEPES , 50 mM NaCl, 1 mM DTT, 50% glycerol, 0.1 mM EDTA, pH 8.0.

Example 4

This example shows that F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 helicase (SEQ ID NO: 1 with mutations C172V/C217A/D284C/S589C/C594A and bismaleimide- PEG3 linkage) to control the movement of entire DNA strands through a single MspA nanopore (SEQ ID NO: 8).

1. Materials and methods

Preparation of DNA construct A as shown in Figure 2: SEQ ID NO: 3 with the 5' end linked to 4 iSpC3 spacers linked to the 3' end of SEQ ID NO: 4 The 5' end of the construct was ligated to SEQ ID NO: 5, and the SEQ ID NO: 6 region of the construct hybridized to SEQ ID NO: 7 (which had a 3' cholesterol tether).

The prepared DNA construct was pre-prepared with F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 in buffer (10 mM HEPES, pH 8.0, 100 mM NaCl, 5% glycerol, 2 mM DTT) at 25°C. Incubate for 30 minutes.

Electrical measurements were obtained from MspA nanopores embedded in a 1,2-diethanolacyl-glycero-3-cholinephospholipid bilayer. By the Montal-Mueller technique, ~25 μm diameter holes in the PTFE membrane formed a bilayer, separating two ~100 μL buffer solutions. All experiments were performed in the described buffer. Single-channel current is measured using an amplifier equipped with a digitizer. The Ag/AgCl electrodes were connected into the buffer such that the cis compartment was connected to the ground of the amplifier and the trans compartment was connected to the active electrode.

After the bilayer achieved a single well, DNA polynucleotides and F8813 helicase were added to 70 μL of buffer in the cis compartment of the electrophysiology chamber to prime the helicase-DNA complex in the nanopore capture. Helicase ATPase activity was activated as needed by addition of divalent metal (5 mM _MgCl2 ) and NTP (2.86 [mu]M ATP) to the cis compartment. Experiments were carried out at a constant potential of +180 mV.

2. Results and Discussion

The results show that the DNA construct is mobilized by F8813 helicase-controlled DNA, and the results of F8813 helicase-controlled DNA mobilization are shown in FIG. 4 . The DNA movement controlled by the F8813 helicase was 24 seconds long and corresponded to the translocation of the DNA construct of nearly 200 bp through the nanopore. Among them, Figure 5 shows an enlarged view of a partial region of DNA movement controlled by the F8813 helicase.

Example 5

This example shows how a DNA-containing leader is linked to RNA to facilitate loading of a DNA helicase, namely F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (with mutation C172V/C217A/D284C/S589C/ SEQ ID NO: 1 of C594A linked to bismaleimide-PEG3) and then observed that helicases control the movement of RNA through the nanopore. 0.28kb RNA was obtained by in vitro transcription. The DNA-containing leader region is ligated to the 3' end of the RNA. The F8813 helicase was then loaded into the DNA ligation site in the leader and the substrate was analyzed through the nanopore.

1. Materials and methods

Preparation of DNA-RNA construct B as shown in Figure 8: SEQ ID NO: 13 with 3' end linked to 20 iSpC3 spacers and its 5' end linked to 4 iSpC3 spacers linked to SEQ ID NO : the 3' end of SEQ ID NO: 14, the 5' end of this SEQ ID NO: 14 is linked by annealing to the RNA single strand SEQ ID NO: 15, the SEQ ID NO: 16 region of the construct is identical to SEQ ID NO: 7 (which has 3 'cholesterol tether) hybridization.

Construct B was then purified using VAHTS RNA Clean Beads at a ratio of 1.8 μL of beads per μL of sample. The purified construct B was reverse transcribed using the SuperScript III kit.

The mixture was then purified using VAHTS RNA Clean Beads at a ratio of 1 μL of beads per μL of sample.

DNA-RNA construct B in buffer (in 50 mM NaCl, 10 mM Tris pH 7.5) was combined with F8813_C172V/C217A/D284C/S589C/C594A-double in buffer (50 mM KCl, 10 mM HEPES, pH 8.0) Maleimide PEG3 was pre-incubated for 30 min at room temperature. Buffer (10 mM HEPES, 600 mM KCl, pH 8.0, 3 mM MgCl ₂ ) and ATP were then added to the premix.

Electrical measurements were obtained from a single MspA nanopore embedded in a block copolymer in buffer (10 mM HEPES, 400 mM KCl, pH 8.0) at room temperature. After achieving single pore insertion into the block copolymer, F8813 helicase (F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 (1 nM final concentration)), DNA (0.3 nM final concentration) , a premix of fuel (ATP 3mM final concentration) was added to the single nanopore experimental system. Each experiment was performed at a holding potential of 180 mV for 2 hours and monitored F8813 helicase-controlled RNA movement.

2. Results

For DNA-RNA construct B, the RNA movement controlled by F8813 helicase was observed, and the results of the RNA movement controlled by F8813_C172V/C217A/D284C/S589C/C594A-bismaleimide PEG3 are shown in Figures 9-10.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner under the condition of no contradiction. In order to avoid unnecessary repetition, the present invention has The combination method will not be specified otherwise.

Claims

A modified F8813 helicase, characterized in that the F8813 helicase comprises a polynucleotide binding domain, the F8813 helicase comprises a variant of SEQ ID NO: 1, and the SEQ ID Variants of NO: 1 include the introduction of at least one cysteine residue and/or at least one unnatural amino acid in the 2A domain, Ratchet domain and/or HLH domain of SEQ ID NO: 1 to reduce multinucleation The size of the opening of the nucleotide binding domain in which the F8813 helicase retains its ability to control movement of polynucleotides.
The F8813 helicase of claim 1, wherein the variant of SEQ ID NO: 1 is included in positions E250 to H310 of the 2A domain of SEQ ID NO: 1, and/or the Ratchet domain At least one cysteine residue and/or at least one unnatural amino acid has been introduced into positions L580 to G600 of the HLH domain, and/or positions D680 to I700 of the HLH domain.
The F8813 helicase according to claim 1 or 2, wherein the variant of SEQ ID NO: 1 comprises the introduction of at least one cysteine at the D284 and/or S589 position of SEQ ID NO: 1 residues and/or at least one unnatural amino acid.
The F8813 helicase according to any one of claims 1-3, wherein the variant of SEQ ID NO: 1 is further included in S272, A273, E274, E281, D284, E285 of SEQ ID NO: 1 , L287, E288, N289, S290, E291, D293, T294, A300, R303, T314, T315, P316, L317, R318, L320, E322, R326, G328, S589, K691, D694, R695, or K698 At least one cysteine residue and/or at least one unnatural amino acid is introduced at one or more positions.
The F8813 helicase according to any one of claims 1-4, wherein the amino acid sequence of the F8813 helicase is SEQ ID NO: 1 or a variant thereof, or the amino acid shown in SEQ ID NO: 1 or a variant thereof The sequence has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 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%, or at least 99.9% homology, and have the ability to control polynucleotide movement.
The F8813 helicase according to any one of claims 1-5, wherein the introduced cysteine and the cysteine are connected to each other, and the introduced unnatural amino acid and the unnatural amino acid are mutually connected Linking, the introduced cysteine is linked to the unnatural amino acid, the introduced cysteine is linked to the natural amino acid, or the introduced unnatural amino acid is linked to the natural amino acid.
The F8813 helicase according to any one of claims 1-6, wherein the variant of SEQ ID NO: 1 further comprises that at least one or more cysteines of SEQ ID NO: 1 are substituted; Preferably, cysteine is replaced by alanine, serine or valine.
The F8813 helicase according to claim 7, wherein the substituted one or more cysteine is one of C172, C217, C246, C256, C301, C469, C527 or C594 or A combination of two or more.
The F8813 helicase according to any one of claims 1-8, wherein the F8813 helicase comprises removing the C-terminal HLH domain of the F8813 helicase, preferably removing the C-terminal HLH domain from A644 to Y729 amino acid sequence.
The F8813 helicase according to any one of claims 1-9, wherein the unnatural amino acid is selected from 4-azido-L-phenylalanine (Faz), 4-acetyl-L -Phenylalanine, 3-Acetyl-L-Phenylalanine, 4-Acetoacetyl-L-Phenylalanine, O-Allyl-L-Tyrosine, 3-(Phenylselenyl )-L-alanine, O-2-propyn-1-yl-L-tyrosine, 4(dihydroxyboryl)-L-phenylalanine, 4-[(ethylsulfanyl) Carbonyl]-L-phenylalanine, (2S)-2-amino-3-{4-[(propan-2-ylsulfanyl)carbonyl]phenyl}propionic acid, (2S)-2-amino- 3-{4-[(2-Amino-3-sulfanylpropionyl)amino]phenyl}propionic acid, O-methyl-L-tyrosine, 4-amino-L-phenylalanine, 4 -Cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L -Phenylalanine, O-(trifluoromethyl)tyrosine, 4-nitro-L-phenylalanine, 3-hydroxy-L-tyrosine, 3-amino-L-tyrosine, 3 -Iodo-L-tyrosine, 4-isopropyl-L-phenylalanine, 3-(2-naphthyl)-L-alanine, 4-phenyl-L-phenylalanine, ( 2S)-2-amino-3-(naphthalen-2-ylamino)propionic acid, 6-(methylsulfanyl)norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-Hydroxy-3-(4-hydroxyphenyl)propionic acid, (2R)-2-aminocaprylate 3-(2,2′-dipyridin-5-yl)-D-alanine , 2-amino-3-(8-hydroxy-3-quinolinyl)propionic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, ( 2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl]propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-Dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl} Amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine, 2-nitrophenylalanine, 4-[(E)-phenyldiazenyl]-L-phenylpropane Amino acid, 4-[3-(trifluoromethyl)-3H-diaziridinyl-3-yl]-D-phenylalanine, 2-amino-3-[[[5-(dimethylamino) -1-Naphthyl]sulfonylamino]propionic acid, (2S)-2-amino 4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanoic acid, (2S)-3-[ (6-Acetylnaphthalen-2-yl)amino]-2-aminopropionic acid, 4(carboxymethyl)phenylalanine, 3-nitro-L-tyrosine, O-thio-L-tyrosine Amino acid, (2R)-6-acetamido-2-aminocaproate, 1-methylhistidine, 2-aminononanoic acid, 2-aminodecanoic acid, L-homocysteine, 5- Sulfanyl norvaline, 6-sulfanyl-L-norleucine, 5-(methyl sulfide alkyl)-L-norvaline, N6-{[(2R,3R)-3-methyl-3,4-dihydro-2H-pyrrole 2-yl]carbonyl}-L-lysine, N6 -[(benzyloxy)carbonyl]lysine, (2S)-2-amino-6-[(cyclopentylcarbonyl)amino]hexanoic acid, N6-[(cyclopentyloxy)carbonyl]-L -Lysine, (2S)-2-amino-6-{[(2R)-tetrahydrofuran-2-ylcarbonyl]amino}hexanoic acid, (2S)-2-amino-8-[(2R,3S)- 3-Ethynyltetrahydrofuran-2-yl]-8-oxyoctanoic acid, N6-(tert-butoxycarbonyl)-L-lysine, (2S)-2-hydroxy-6-({[(2-methyl (2-Propyloxy)oxy]carbonyl}amino)hexanoic acid, N6-[(allyloxy)carbonyl]lysine, (2S)-2-amino-6-({[(2-azido Benzyl)oxy]carbonyl}amino)hexanoic acid, N6L-prolyl-L-lysine, (2S)-2-amino-6-{[(prop-2-yn-1-yloxy) Carbonyl]amino}hexanoic acid or N6-[(2azidoethoxy)carbonyl]-L-lysine.
A kind of construct is characterized in that, described construct comprises at least one F8813 helicase described in any one of claim 1-10.
The construct of claim 11, wherein the construct further comprises a polynucleotide binding moiety.
A nucleic acid, characterized in that the nucleic acid encodes the F8813 helicase described in any one of claims 1-10 or the construct described in any one of claims 11-12.
An expression vector, characterized in that the expression vector comprises the nucleic acid of claim 13 .
A host cell, wherein the host cell comprises the nucleic acid of claim 13 or the expression vector of claim 14.
A method for preparing the F8813 helicase described in any one of claims 1-10, characterized in that SEQ ID NO: 1 is provided, and at least one cysteine residue and/or at least one cysteine residue are introduced in SEQ ID NO: 1 A variant of SEQ ID NO: 1 was obtained with an unnatural amino acid to reduce the opening size of the polynucleotide binding domain of the F8813 helicase, wherein the F8813 helicase retains its ability to control polynucleotide movement.
A method for preparing the F8813 helicase according to any one of claims 1 to 10, wherein the host cell according to claim 15 is cultured and induced to express, and the F8813 helicase is obtained by purification.
A method for regulating the opening size of a polynucleotide binding domain of a helicase, wherein the method comprises adding the F8813 helicase according to any one of claims 1-10 or any one of claims 11-12 The construct is contacted with the polynucleotide, preferably to reduce the size of the opening in the binding domain of the polynucleotide.
A method for controlling the movement of a polynucleotide, wherein the method comprises combining the F8813 helicase described in any of claims 1-10 or the construct described in any of claims 11-12 with a polynucleoside acid contact.
A method for characterizing a target polynucleotide, wherein the method comprises:

1) contacting the F8813 helicase of any one of claims 1-10 or the construct of any one of claims 11 to 12 with the target polynucleotide and the pore, so that the F8813 helicase or the construct controls the target movement of the polynucleotide through the pore;

and II) obtaining one or more characteristics of the interaction of nucleotides in the target polynucleotide with the pore to characterize the target polynucleotide.
The method of claim 20, further comprising the step of applying a potential difference across the pore in contact with the helicase or construct, and the target polynucleotide; preferably, the The wells are selected from biological wells, solid state wells, or biological and solid state hybrid wells.
The method according to claim 20 or 21, wherein the target polynucleotide is single-stranded, double-stranded or at least partially double-stranded, and the target polynucleotide is DNA or RNA.
The method according to any one of claims 20-22, wherein the one or more features are selected from the source, length, identity, sequence, secondary structure of the target polynucleotide or whether the target polynucleotide is is modified; preferably, the one or more features are carried out by electrical and/or optical measurements.
A product for characterizing a target polynucleotide, characterized in that the product comprises the F8813 helicase described in any one of claims 1-10, the construct described in any one of claims 11-12, the claim 13 The nucleic acid, the expression vector of claim 14 or the host cell of claim 15.
The product of claim 24, wherein the product is selected from a kit, a device or a sensor.
The F8813 helicase described in any one of claims 1-10, the construct described in any one of claims 11-12, the nucleic acid described in claim 13, the expression vector described in claim 14, the described in claim 15 Use of the host cell of any one of claims 24-25 or the product of any one of claims 24-25 in characterizing a polynucleotide of interest or controlling the movement of a polynucleotide of interest through a pore.