CN112501267A - RNA exonuclease-nanopore complex and preparation method and application thereof - Google Patents

Abstract

An exonuclease-nanopore complex and its preparation method and application. The application is an RNA sequencing method, which includes the following steps: using an RNA exonuclease-nanopore complex, firstly using an RNA exonuclease to start the sequencing sample from one end, cutting out single nucleotides in sequence and passing through the nanopore, and then using the RNA exonuclease The characteristics of nucleotides passing through the nanopore are used to determine basic nucleotides and modified nucleotides; if the determination result is basic nucleotides, the type and sequence of AUCG are recorded; if the determination result is modified nucleotides, record Its position, and the type of its chemical modification is detected by other spectral features; through the above process, the sequence information of the nucleotide and its chemical modification of the sequenced sample is obtained, and the direct reading of the RNA sequence is realized. The RNA exonuclease-nanopore complex includes: nanopore structure; RNA exonuclease immobilized in the nanopore structure. The RNA exonuclease-nanopore complex can be used for direct RNA detection, and the sequencing speed is fast and can ensure that various modified nucleotides can be detected.

Description

RNA exonuclease-nanopore complex and preparation method and application thereof

Technical Field

The invention relates to the technical field of gene sequencing, in particular to an RNA exonuclease-nanopore complex and a preparation method and application thereof.

Background

RNA is an important macromolecular substance composing life, and is an important substance basis for the biological functions of cells and living bodies. RNA is a single-stranded, long-chain macromolecule composed of A, U, C, G four basic nucleotides (nt). In addition, A, U, C, G four nucleotides can be catalyzed by enzyme to form nucleotides with different chemical groups for modification, and the modifications also have important biological functions. Biomedical research requires knowledge of the nucleotide sequence of each single RNA molecule in a sample, and which chemical modifications have been made to the nucleotides at which positions, i.e. direct measurement of the sequence of the single RNA molecule (including modified nucleotides). In natural RNA molecules, basic nucleotides are mainly used, and the proportion of modified nucleotides is only about 0.1%, but the variety is wide, and more than 170 kinds of modified nucleotides with different chemical structures are known, and the accurate distinction at a single molecule level is quite challenging.

The existing RNA direct detection technology comprises nanopore sequencing and spectrum detection. The nanopore is high in sequencing speed, about 1000 nt/s, but can only distinguish A, U, C, G four basic nucleotides and several kinds of modified nucleotides with limited types; raman spectroscopy and other spectroscopic detection can distinguish hundreds of modified nucleotides, but the speed is slow, the integration time for detecting single nucleotide is usually more than 1 second, and the parallel detection at the single molecule level is difficult to realize. Even if a specific optical structure design is applied, the Raman spectrum signal is gained, and the integration time is shortened to 100ms (CN110628599A), more than 100 parallel detections are difficult to realize. Thus, for a conventional single-cell RNA sequencing sample, it often contains 1X 10⁹Individual nucleotides, which need to be detected spectroscopically alone>The time of 100 days is far from meeting the requirement of practical application.

Disclosure of Invention

In view of the above, the present invention provides an exonuclease-nanopore complex, a preparation method and an application thereof, and the exonuclease-nanopore complex provided by the present invention can be used for RNA direct detection, has a high sequencing speed, and can ensure that various modified nucleotides can be determined.

The invention provides an RNA exonuclease-nanopore complex, comprising:

a nanopore structure;

an exonuclease immobilized within the nanopore structure.

The rate of the RNA exonuclease hydrolysis of basic nucleotides (AUCG) and most of modified nucleotide monomers is obviously different.

Preferably, the nanopore structure is a protein nanopore; the protein nanopore performs fusion expression and self-assembly on the RNA exonuclease through a fusion protein technology to form a couplet of the RNA exonuclease and the protein nanopore.

Preferably, the protein nanopore is an alpha-hemolysin protein nanopore for modifying beta-cyclodextrin, MspA, CsgG or other nanopore which can be used for sequencing.

Preferably, the nanopore structure is a solid-state nanopore; the solid-state nanopore leads and modifies RNA exonuclease near the solid-state nanopore by a click chemistry method to form a coupling structure of the RNA exonuclease and the solid-state nanopore.

Preferably, the exonuclease comprises an exonuclease from a prokaryote or a mutant thereof.

The invention also provides a preparation method of the RNA exonuclease-nanopore complex, which comprises the following steps:

a) constructing a solid supporting structure of the RNA exonuclease nanopore complex,

b) embedding the RNA exonuclease-protein nanopore fusion protein in the solid support structure to form an RNA exonuclease-nanopore complex; or preparing a needle tip nanopore in a solid support structure, and then fixing the RNA exonuclease near the edge of the solid support structure to form an RNA exonuclease-nanopore complex.

The invention also provides a method for sequencing RNA, which comprises the following steps:

according to the RNA exonuclease-nanopore complex, firstly, RNA exonuclease is utilized to cut single nucleotides from one end of a sequencing sample in sequence and enable the single nucleotides to pass through a nanopore, and then basic nucleotides and modified nucleotides are judged by utilizing the characteristic that the nucleotides pass through the nanopore;

if the judgment result is the basic nucleotide, recording the type and the sequence of AUCG;

if the judgment result is the modified nucleotide, recording the position of the modified nucleotide, and determining the chemically modified type of the modified nucleotide through the detection of the spectral characteristics;

through the process, the sequence information of the nucleotide of the sequencing sample is obtained, and the direct reading of the RNA sequence is realized.

Preferably, the process of determining the basic nucleotide and the modified nucleotide by using the characteristic that the nucleotide passes through the nanopore specifically comprises the following steps:

high salt solution is poured into two sides of the nanopore, positive and negative electrodes are applied to form transmembrane voltage and ion current crossing the nanopore, and a patch clamp amplification circuit is applied to record the ion current;

nucleotide enters the high-salt solution from the negative electrode side, passes through the nanopore under the driving of an electric field and gushes to the positive electrode;

and recording the transient reduction of the ion current signal when the nucleotide passes through the nanopore as a one-time hole crossing event, and judging whether the nucleotide passing through the nanopore is a basic nucleotide or a modified nucleotide according to the time interval between the time when the nucleotide passes through the nanopore and the previous hole crossing event.

Preferably, a Y-shaped nano-flow channel is designed on one side of the nanopore positive electrode, and a basic nucleotide capture electrode and a modified nucleotide capture electrode are respectively arranged in the two branched channels;

the basic nucleotide capture electrode is opened to drive the mononucleotide to pass through the nanopore and enter the channel where the mononucleotide is located; when the modified nucleotide passes through the nanopore, as the time interval of the cross-pore event is increased, the modified nucleotide capture electrode is started through the feedback circuit design to enable the capture voltage of the modified nucleotide capture electrode to be higher than that of the basic nucleotide capture electrode, and the modified nucleotide is adsorbed to another channel, so that the separation of the modified nucleotide is realized;

after the modified nucleotide enters the branched channel through the nanopore, the capture voltage of the modified nucleotide is rapidly reduced to be lower than that of the basic nucleotide capture electrode, and the subsequent basic nucleotide is ensured to enter the correct channel.

Preferably, the method for detecting the chemically modified species of the modified nucleotide is selected from one or more of single molecule raman spectroscopy, fluorescence spectroscopy, infrared spectroscopy, absorption spectroscopy, reflection spectroscopy, mass spectroscopy, dielectric properties and energy spectroscopy.

The invention provides an RNA exonuclease-nanopore complex and a preparation method and application thereof. The application is a sequencing method of RNA, comprising the following steps: adopting an RNA exonuclease-nanopore complex, firstly, sequentially cutting off single nucleotides from one end of a sequencing sample by using RNA exonuclease and enabling the single nucleotides to pass through a nanopore, and then judging basic nucleotides and modified nucleotides by using the characteristic that the nucleotides pass through the nanopore; if the judgment result is the basic nucleotide, recording the type and the sequence of AUCG; if the judgment result is the modified nucleotide, recording the position of the modified nucleotide, and detecting the chemically modified type of the modified nucleotide; through the process, the sequence information of the nucleotide of the sequencing sample is obtained, and the direct reading of the RNA sequence is realized. The exonuclease-nanopore complex comprises: a nanopore structure; an exonuclease immobilized within the nanopore structure. Compared with the prior art, the RNA exonuclease-nanopore complex provided by the invention can be used for RNA direct measurement, has high sequencing speed and can ensure that various modified nucleotides can be measured.

Drawings

FIG. 1 is a diagram showing the decrease in the rate of hydrolysis of a modified nucleotide by RNase R in the present invention;

FIG. 2 is a schematic diagram of an exonuclease-protein nanopore conjugate provided by an embodiment of the present invention;

FIG. 3 is a schematic diagram of a design of a modified nucleotide sorting nanoflow channel and capture electrode in an embodiment of the invention;

FIG. 4 is a schematic structural diagram of a sequencing unit based on an exonuclease-nanopore coupled protein provided in example 1 of the present invention;

FIG. 5 is a schematic structural diagram of a modularly assembled nanopore device provided in example 1 of the present invention;

FIG. 6 is a schematic diagram of the structure of a complex of exonuclease-complexed solid state nanopore structure provided in example 2 of the present invention;

FIG. 7 is a schematic diagram of the exonuclease based nucleic acid sequencing system provided in example 2 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides an RNA exonuclease-nanopore complex, comprising:

a nanopore structure;

an exonuclease immobilized within the nanopore structure.

In the present invention, the exonuclease-nanopore complex comprises a nanopore structure and an exonuclease; the RNA exonuclease is immobilized within the nanopore structure.

In a preferred embodiment of the invention, the nanopore structure is a protein nanopore; the protein nanopore performs fusion expression and self-assembly on the RNA exonuclease through a fusion protein technology to form a couplet of the RNA exonuclease and the protein nanopore. In the present invention, the protein nanopore is typically composed of 8-12 monomers, forming a porous protein channel; the protein nanopore may be an alpha-hemolysin protein, MspA, CsgG, preferably an alpha-hemolysin protein nanopore that modifies beta-cyclodextrin. The alpha-hemolysin protein nanopore of the modified beta-cyclodextrin, which can be used for nucleic acid sequencing, has single nucleotide resolution, can identify single basic nucleotide passing through a pore channel of the nanopore, and has an accuracy rate of distinguishing A, U, C, G four nucleotides up to 99% (Ayub M, et al. Nano Lett.2013,12: 6144-50.). At present, the frequency of detecting the cross-nanopore ionic current by using the patch clamp current can reach 100kHz, the detection frequency of a sequencing chip for detecting the cross-nanopore ionic current in a parallelized manner by using an integrated circuit also reaches 10kHz, the passing nucleotides at 1000 nt/s can be accurately detected, and the requirements of the invention on a nanopore detection part are met.

In the present invention, the RNA exonuclease is capable of sequentially hydrolyzing a single-stranded nucleic acid in an RNA molecule from one end to a single nucleotide; for a natural RNA sample, RNA exonuclease is needed, but natural DNA exonuclease or DNA exonuclease which is artificially modified and can take RNA as a substrate can also be used; among them, there are many kinds of natural exonuclease, each organism encodes many kinds of exonuclease, the exonuclease preferably includes exonuclease or mutant thereof of prokaryotes and eukaryotes; exonucleases from prokaryotes or mutants thereof are more commonly used because they are more readily expressed in vitro to obtain active enzymes; the prokaryotic RNA exonuclease comprises protein families such as PNP, RNase II, RNaseR and the like.

In a preferred embodiment of the invention, the exonuclease is RNase R; RNaseR has 3 '→ 5' exorna activity and is capable of degrading almost all linear RNAs, including rRNA and tRNA with complex modified nucleotides; RNaseR consists of a single monomer, comprises two structural domains of hydrolysis and unwinding, has unwinding activity, and can open the secondary structure of single-stranded RNA and hydrolyze the single-stranded RNA into single nucleotides from the tail end in turn. The RNaseR protein (the amino acid sequence of which is shown in SEQ ID NO: 1) from the psychrophile Antarctica (Psychrobacters sp.) is expressed in vitro, has the characteristic of high exoactivity under the conditions of room temperature and high salt, and is more suitable for the application of the invention. The rate of hydrolysis of different nucleotides by exonuclease varies, and usually, hydrolysis is fast when hydrolyzing common nucleotides such as A, U, C, G, and hydrolysis is fast when hydrolyzing modified nucleotides, such as: 170 residual seeds such as m6A, 5mC, 5hmC, m1A and the like, and the speed is obviously slower; the speed difference can be used for distinguishing the basic nucleotide with high content and the modified nucleotide with low content in the RNA sample, and then the types of the nucleotides are judged by respectively using a nanopore detection device with high speed but few types of the identified nucleotides and a spectrum detection device with more types of the identified nucleotides but low speed. The rate of RNaseR hydrolysis of the common RNA strand consisting of the basic nucleotides is generally 50-300 nt/sec (Lee G, et al. science 2012,336(6089): 1726-), (Fazal FM, et al. Proc Natl Acad Sci USA 2015,112(49): 15101-); the present invention compares the rate of RNase R hydrolysis of a base nucleotide and a modified nucleotide from Psychrobacter sp. if two consecutive nucleotides A in an RNA strand consisting of the base nucleotide are replaced with a modified nucleotide m6A, the rate of RNase hydrolysis is significantly slowed down and significant residence occurs at the modified nucleotide (FIG. 1); thus, differences in the rate at which RNaseR hydrolyzes modified nucleotides can be used to sort modified nucleotides.

The present invention is not limited to the fusion protein technology, and the technical scheme known to those skilled in the art can be adopted. The invention applies the fusion protein technology to fuse and express the RNA exonuclease and the protein nanopore to form a protein monomer with the upper section of exonuclease and the lower section of nanopore monomer linked by a hinge (5-20 amino acids), and the protein monomer is mixed with the single nanopore protein monomer to self-assemble a couplet of the RNA exonuclease and the protein nanopore, as shown in figure 2.

In another preferred embodiment of the present invention, the nanopore structure is a solid-state nanopore; the solid-state nanopore leads and modifies RNA exonuclease near the solid-state nanopore by a click chemistry method to form a coupling structure of the RNA exonuclease and the solid-state nanopore. The preparation method of the solid-state nanopore is not particularly limited by the invention, and the technical scheme for constructing the composite solid-state nanopore support structure, which is well known to the technical personnel in the field, can be adopted (refer to patents: CN110628598A, CN 110628597A). Meanwhile, with the gradual improvement of the processing technology of the solid-state nanopore, the accurate identification of the basic nucleotide can be realized, so that the function same as that of the protein nanopore in the technical scheme is realized and the protein nanopore is used for the invention.

In the present invention, the preparation of the exonuclease is the same as that in the above technical scheme, and is not described herein again.

In the invention, the purpose of guiding and modifying the RNA exonuclease by a click chemistry method is to couple the RNA exonuclease to the edge of the solid-state nanopore, and in the preferred embodiment of the invention, the RNA exonuclease is guided and modified on the surface of one side of the lower cavity of the composite solid-state nanopore fluid by using the click chemistry method; the optional method is as follows: mutating certain outer amino acid near a hydrolytic nucleotide outlet of the RNA exonuclease into cysteine, and forming a disulfide bond with a thiol modified at the edge of the solid nanopore, so that the RNA exonuclease is fixed at the edge of the solid nanopore.

The invention also provides a preparation method of the RNA exonuclease-nanopore complex, which comprises the following steps:

constructing a nanopore structure, and then fixing the RNA exonuclease in the nanopore structure to form an RNA exonuclease-nanopore complex.

The preparation method provided by the invention is a preparation method for preparing solid structures for supporting the exonuclease-protein nanopore and modifying nucleotide capture respectively aiming at different nanopore structures, and then embedding the exonuclease-protein nanopore coupled complex into a lipid bilayer on the surface of the solid structure to finally form the protein-solid composite nanopore, and a preparation method for firstly constructing the composite solid nanopore support structure and then fixing the exonuclease in the composite solid nanopore.

The invention also provides a method for sequencing RNA, which comprises the following steps:

according to the RNA exonuclease-nanopore complex, firstly, RNA exonuclease is utilized to cut single nucleotides from one end of a sequencing sample in sequence and enable the single nucleotides to pass through a nanopore, and then basic nucleotides and modified nucleotides are judged by utilizing the characteristic that the nucleotides pass through the nanopore;

if the judgment result is the basic nucleotide, recording the type and the sequence of AUCG;

if the judgment result is the modified nucleotide, recording the position of the modified nucleotide, and detecting the chemically modified type of the modified nucleotide;

through the process, the sequence information of the nucleotide of the sequencing sample is obtained, and the direct reading of the RNA sequence is realized.

In the invention, the RNA exonuclease and the nanopore in the RNA exonuclease-nanopore complex are tightly coupled together, so that the sequence of nucleotides passing through the nanopore is not disturbed; on this basis, the RNA exonuclease sequentially cleaves nucleotides from one end of the nucleic acid strand, which further pass through the nanopore in the order of the cleavage, thereby being able to reduce the order of these nucleotides in the sample nucleic acid strand from the nanopore signal, i.e. sequencing.

In the present invention, the sequencing sample includes, but is not limited to, various natural RNAs extracted from animals, plants, microorganisms, environments, cultures, etc., or enriched mRNAs, tRNAs, rRNAs, miRNAs, circRNAs, and other non-coding RNAs, or artificially synthesized RNAs, or mixtures thereof. In addition, for the kind and position of modification requiring fine measurement of chemically modified deoxynucleotides in DNA, the method of the present invention may also be used, requiring only the exchange of an RNA exonuclease for a DNA exonuclease.

Firstly, sequentially cutting off single nucleotide from one end of a sequencing sample by using RNA exonuclease and enabling the single nucleotide to pass through a nanopore; preferably, the single nucleotides are hydrolyzed one by one from the 3' end of the RNA, and the nucleotides hydrolyzed by the exonuclease are sequentially passed through the nanopore designed next to it.

In the present invention, the process of determining the base nucleotide and the modified nucleotide by using the characteristic that the nucleotide passes through the nanopore is preferably embodied as follows:

high salt solution is poured into two sides of the nanopore, positive and negative electrodes are applied to form transmembrane voltage and ion current crossing the nanopore, and a patch clamp amplification circuit is applied to record the ion current;

nucleotide enters the high-salt solution from the negative electrode side, passes through the nanopore under the driving of an electric field and gushes to the positive electrode;

and recording the transient reduction of the ion current signal when the nucleotide passes through the nanopore as a one-time hole crossing event, and judging whether the nucleotide passing through the nanopore is a basic nucleotide or a modified nucleotide according to the time interval between the time when the nucleotide passes through the nanopore and the previous hole crossing event.

In the invention, when nucleotide passes through a nanopore, an ion current signal is temporarily reduced due to the fact that a spatial effect blocks the ion current across the nanopore, and the ion current signal is recorded as a primary hole-crossing event, the basic nucleotide has high hydrolysis speed, the time interval between the basic nucleotide and the previous hole-crossing event is short (calculated as 3-20 ms according to the hydrolysis speed of RNaseR) when the basic nucleotide passes through the nanopore, and the time interval between the basic nucleotide and the previous hole-crossing event is longer due to the slow hydrolysis speed of the modified nucleotide, so that whether the nucleotide passing through the nanopore at this time is the basic or the modified nucleotide can be judged according to the time interval between the basic nucleotide and the previous hole-crossing event.

In the invention, a Y-shaped nano-flow channel is designed on one side of the nanopore positive electrode, and a basic nucleotide capture electrode and a modified nucleotide capture electrode are respectively arranged in two branched channels (shown in figure 3);

the basic nucleotide capture electrode is opened to drive the mononucleotide to pass through the nanopore and enter the channel where the mononucleotide is located; when the modified nucleotide passes through the nanopore, the modified nucleotide capture electrode is started and closed through the feedback circuit design or the voltage of the modified nucleotide capture electrode is higher than the capture voltage of the base nucleotide due to the increase of the time interval, and the modified nucleotide is adsorbed to another channel, so that the separation of the modified nucleotide is realized;

after the modified nucleotide enters the branched channel through the nanopore, the capture voltage of the modified nucleotide is rapidly reduced, and the basic nucleotide capture electrode is opened, so that the capture voltage of the basic nucleotide is higher than that of the modified nucleotide, and the subsequent basic nucleotide is ensured to enter the correct channel.

The invention realizes the sorting of the modified nucleotides by adopting the process that the feedback circuit triggers the capture electrode to apply positive voltage. In the present invention, the time interval when the modified nucleotide passes through the nanopore is preferably > 10ms, and the time interval when the base nucleotide passes through the nanopore is preferably less than 10 ms.

In the present invention, if the determination result is a modified nucleotide, the position thereof is recorded, and the kind of chemical modification thereof is determined using a spectroscopic detection signal; the process of detecting the chemically modified species thereof preferably further comprises:

and adsorbing the modified nucleotide to a spectral detection area by using a capture electrode, and performing spectral detection to obtain the chemically modified type of the modified nucleotide.

In the present invention, the method for detecting the chemically modified species of the modified nucleotide is preferably selected from one or more of monomolecular raman spectroscopy, fluorescence spectroscopy, infrared spectroscopy, absorption spectroscopy, reflection spectroscopy, mass spectroscopy and energy spectroscopy; the chemical structure of the modified nucleotide can be judged by the spectral characteristics of the chemical molecules. In a preferred embodiment of the present invention, the chemical structure of the modified nucleotide is determined by single-molecule Raman spectroscopy (refer to patent: CN 110628599A); the single molecule spectrum detection has a hot spot effect, namely, the molecule to be detected is positioned in a hot spot area, and enough signals can be obtained. In the embodiment, the modified nucleotide can be captured to the hot spot region detected by the Raman spectrum by using the metal structure of the hot spot region and the capture electrode of the modified nucleotide, so that the sensitivity of the spectrum detection is improved.

Through the process, the sequence information of the nucleotides (including modified nucleotides) of the sequencing sample can be obtained, and the direct reading of the RNA sequences (including modified nucleotides) is realized.

The invention provides an RNA exonuclease-nanopore complex and a preparation method and application thereof. The application is a sequencing method of RNA, comprising the following steps: adopting an RNA exonuclease-nanopore complex, firstly, sequentially cutting off single nucleotides from one end of a sequencing sample by using RNA exonuclease and enabling the single nucleotides to pass through a nanopore, and then judging basic nucleotides and modified nucleotides by using the characteristic that the nucleotides pass through the nanopore; if the judgment result is the basic nucleotide, recording the type and the sequence of AUCG; if the judgment result is the modified nucleotide, recording the position of the modified nucleotide, and detecting the chemically modified type of the modified nucleotide; through the process, the sequence information of the nucleotide of the sequencing sample is obtained, and the direct reading of the RNA sequence is realized. The exonuclease-nanopore complex comprises: a nanopore structure; an exonuclease immobilized within the nanopore structure. Compared with the prior art, the RNA exonuclease-nanopore complex provided by the invention can be used for RNA direct measurement, has high sequencing speed and can ensure that various modified nucleotides can be measured.

To further illustrate the present invention, the following examples are provided for illustration.

Example 1 exonuclease-protein nanopore coupled Structure

(1) Forming a protein nanopore support and nucleotide capture solid state structure:

firstly, forming a sacrificial layer containing a silicon compound on a silicon wafer substrate, and then forming a base layer on the sacrificial layer containing the silicon compound;

spin-coating photoresist on the surface of the silicon layer, manufacturing a mask plate by electron beam exposure, and forming a square silicon hole substrate with the edge width of 1-1000 microns (preferably 500 microns) by using Reactive Ion Etching (RIE);

forming a nano-pore structure on the silicon pore substrate by using potassium hydroxide or tetramethylammonium hydroxide through wet etching to form a Raman spectrum detection solid structure;

fixing the top of the Raman spectrum detection solid structure on a flexible substrate (or other substrates), rotationally coating photoresist on the surface of a silicon layer at the bottom of a substrate layer of the Raman spectrum detection solid structure, manufacturing a mask plate by electron beam exposure, and forming a circular silicon hole substrate with the diameter of 1-1000 microns (preferably 100 microns) by using Reactive Ion Etching (RIE).

(2) Forming an exonuclease-protein nanopore coupled complex:

firstly, in order to reduce the influence of steric hindrance on the formation and activity of the space structures of two monomeric proteins, the invention designs a short peptide chain GGGGSEAAAKEAAAKHHHHHH with the length of 21 amino acids to link the two proteins so as to better fix the space structures of the two monomers;

preparing a fusion protein gene by adopting a gene synthesis strategy, and synthesizing an 8 × His sequence label, exonuclease (removing stop codon), a link peptide and a protein nanopore monomer gene (reserving stop codon) gene from the N end;

carrying out homologous recombination on the fusion gene between the NdeI and Hind III restriction sites of a prokaryotic expression vector PET26b vector, transforming the Escherichia coli DH5 alpha, carrying out PCR (polymerase chain reaction) and sequencing screening, and identifying positive clone;

extracting fusion protein PET26b expression plasmid, transforming BL21(DE3) pLysS competent cell, and plate cloning and screening; selecting positive clones, inoculating the positive clones to 10ml LB culture medium containing kan and Cm antibiotics, and culturing overnight; the next day, according to 1: culturing 100 ml LB culture medium containing Kan and Cm at 25 deg.C and 200rpm to OD600 value of 0.3-0.5, adding IPTG with final concentration of 0.2-0.4 μ M, inducing overnight at 16 deg.C, centrifuging, and collecting thallus;

adding 25ml lysis buffer 20mM Tris-HCl (pH 8.0), 500mM NaCl, 1mM DTT, 0.1mM EDTA, homogenizing by a low-temperature ultrahigh-pressure continuous flow cell crusher, and centrifuging at 12000rpm at 4 ℃ for 15 min;

sixthly, adding imidazole with the final concentration of 10mM into the supernatant, loading the supernatant to a Ni-NTA Resin column for purification, and after balancing 5 volumes by using the buffer solution, eluting the supernatant by using 100mM imidazole 20mM Tris-HCl (pH 8.0);

seventhly, after the eluent is loaded on a Resource Q column and balanced, the eluent is linearly eluted by 100-350mM NaCl 20mM Tris-HCl (pH 8.0) buffer solution and is collected in different time;

subjecting the collected liquid to superdex200 molecular sieve column for further purification and desalination, and collecting protein peak by ultraviolet monitoring; the purity of PAGE identification needs to be more than 90%;

ninthly, mixing the fusion protein and the nano-pore monomer protein expressed by the same method according to a molar ratio of 1: (1-30) (preferably 1: 8-12), and self-assembling into the exonuclease-protein nanopore coupled complex.

(3) Forming a sequencing unit based on the exonuclease-nanopore coupled protein:

perfusing electrophoresis liquid at the bottom of a support solid structure to cover the surface of a circular silicon hole, and then adding diphytanoyl phosphatidylcholine (DPhPC) (or other high molecular polymers capable of forming a lipid bilayer) into the electrophoresis liquid;

absorbing the liquid on the surface of the silicon hole until the liquid just leaks out of the upper edge of the silicon hole, and forming a lipid bilayer covering the silicon hole on the upper edge of the silicon hole;

supplementing an electrophoresis liquid to completely cover the silicon pores and the lipid bilayer, adding the self-assembled exonuclease-protein nanopore coupled compound obtained in the step (2), and spontaneously embedding the protein nanopore into the lipid bilayer to form a sequencing unit based on the exonuclease-nanopore coupled protein.

The structural schematic diagram of the sequencing unit based on the exonuclease-nanopore coupled protein provided in the embodiment 1 of the invention is shown in fig. 4.

The present invention also provides a modularly assembled nanopore device, as shown in fig. 5; the nanopore device comprises a shell, a fluid lower cavity sealing layer, a protein-solid composite nanopore structure, a fluid lower cavity base, a power supply, a first electrode, a second electrode and a third electrode; the first electrode is embedded and integrated above the substrate with the nanopore structure, the second electrode is embedded and integrated on the side wall of the silicon pore and connected with the lower fluid cavity, the third electrode is positioned at the bottom of the lower fluid cavity, the first electrode and the second electrode are respectively connected with the anode of the power supply, and the third electrode is connected with the cathode of the power supply and used for driving nucleic acid fragments and nucleotide molecules in a solution to be detected (such as a solution containing DNA fragments) to flow to the anode from one side of the cathode through the nanopore by using an electrophoresis technology; the remaining technical details of the nanopore structure are fully set forth above and will not be described further herein.

Example 2 exonuclease-solid nanopore coupled structures

(1) Forming a composite solid state nanopore structure:

firstly, forming a sacrificial layer containing a silicon compound on a silicon wafer substrate, and then forming a base layer on the sacrificial layer containing the silicon compound;

spin-coating photoresist on the surface of the silicon layer, manufacturing a mask plate by electron beam exposure, and forming a square silicon hole substrate with the edge width of 1-1000 microns (preferably 500 microns) by using Reactive Ion Etching (RIE);

forming a nano-pore structure on the silicon pore substrate by using potassium hydroxide or tetramethylammonium hydroxide through wet etching to form a Raman spectrum detection solid structure;

fixing the top of the Raman spectrum detection solid structure on a flexible substrate (or other substrates), forming double-layer crossed silicon nanowires at the bottom of the Raman spectrum detection solid structure to construct a nanopore with a thickness close to zero (refer to patent CN111440855A), and forming a metal layer on one side of a lower cavity of a nanopore fluid by using a sputtering process (or an evaporation process), wherein the metal layer is one of copper, silver, gold, zinc, mercury, cadmium, cobalt, nickel or aluminum, and the embodiment is gold.

(2) Immobilizing exonuclease to the composite solid nanopore:

using a gene engineering method to carry out site-directed mutagenesis on special amino acid residues on the surface of an exonuclease protein and replace the special amino acid residues into cysteine; this example uses RNase R as the exonuclease;

secondly, connecting one end of the nucleic acid fragment to be detected with single-stranded oligonucleotide which can be identified by exonuclease, wherein the length of the single-stranded oligonucleotide is 5-100 nt; the single-stranded oligonucleotide described in this example was poly (A) having a length of 8 to 50nt, which was easily bound to RNase R.

Driving single-stranded oligonucleotide by using an electrophoresis method, drawing exonuclease protein into the bottom recess of the lower cavity of the composite nanopore fluid by using the single-stranded oligonucleotide, and stabilizing the exonuclease protein at a position close to the composite nanopore in the lower cavity of the fluid;

and fourthly, combining sulfydryl of cysteine modified on the surface of the exonuclease with the surface of the gold layer to form a gold-sulfur bond, and anchoring the exonuclease protein to a position close to the nanopore in the lower cavity of the composite nanopore fluid through the gold-sulfur bond to form a complex of the exonuclease-composite solid nanopore structure.

The structural schematic diagram of the complex of the exonuclease-composite solid nanopore structure provided in example 2 of the present invention is shown in fig. 6.

The present invention also provides an exonuclease-based nucleic acid sequencing system, as shown in figure 7; the Raman spectrum biological molecule sequencing system comprises a composite nanopore, a patch clamp current detection system, a laser Raman microscope, a spectrum measuring device and a data acquisition and analysis device; the composite nanopore is arranged below the laser Raman microscope.

The nanopore device comprises a shell, a fluidic lower cavity sealing layer, a composite nanopore structure, a fluidic lower cavity base, a power supply, a first electrode, a second electrode and a third electrode; the first electrode is embedded and integrated above the substrate of the nanopore structure, the second electrode is embedded and integrated on the side wall of the silicon pore and connected with the lower fluid cavity, the third electrode is positioned at the bottom of the lower fluid cavity, the first electrode and the second electrode are respectively connected with the anode of the power supply, the third electrode is connected with the cathode of the power supply, and the third electrode is in a disconnected state under normal conditions and is switched on under the regulation and control of a feedback circuit; the composite substrate nanopore structure comprises a first liquid flow channel, a second liquid flow channel, a third liquid flow channel, a fluid upper cavity (above a spectrum nanopore), a nanopore intermediate fluid cavity (between the spectrum nanopore and a needle-tip nanopore), and a fluid lower cavity (below the needle-tip nanopore); the first liquid flow channel is connected with the fluid upper cavity from the outside so as to input or discharge solution to or from the fluid upper cavity; the second liquid flow channel is connected with the fluid middle cavity from the outside so as to input or discharge solution into or out of the fluid middle cavity; the third liquid flow channel is connected with the outside and the fluid lower cavity so as to input or discharge solution in the fluid lower cavity; the fluid cavity sealing layer seals the fluid cavity; the above technical features included in the nanopore device are described in detail above and will not be described herein.

Application examples

(1) The solution containing the biomolecule to be measured enters a fluid lower cavity through a third liquid flow channel and is driven to a needle tip nanopore cavity by spontaneous diffusion or electrophoresis; the probe is captured by the exonuclease in the exonuclease-protein nanopore coupled complex in the embodiment 1, or in the composite nanopore in the embodiment 2, the exonuclease is combined with the biomolecule to be detected before loading, and then the biomolecule is loaded into the lower cavity of the fluid, and then the exonuclease is combined on the surface of the metal layer of the pinpoint nanopore through a click chemical reaction.

(2) Injecting ATP molecules through the third liquid flow channel to supply energy to the exonuclease, hydrolyzing the monomers of the biomolecules from one end of the exonuclease in sequence, and is connected with the first electrode and the second electrode of the patch clamp system to measure the obtained ion current crossing the needle point nanopore through the needle point nanopore under the action of an electric field formed by the first electrode and the second electrode, when a biomolecular monomer (such as a single nucleotide) passes through the needle-tip nanopore, a blocking signal of the ionic current is measured, called a cross-pore event, and when a threshold time is exceeded after the cross-pore event, in a preferred embodiment of the invention, this threshold may be 10ms when RNase R hydrolyzes RNA, and when no cross-pore event has occurred, then the third electrode is activated by the feedback circuit to apply a trapping voltage, which may be from 10 to 200mV, and in a preferred embodiment of the invention, from 30 to 70 mV; and simultaneously closing the second electrode, capturing the monomer passing through the needle tip nanopore to a spectrum nanopore hot spot region by the third electrode, rapidly reducing the voltage of the third electrode to below 10mV after the capturing is finished, simultaneously opening the second electrode, and stopping continuously capturing.

(3) After the spectroscopic detection is completed, a short reverse voltage is applied to the third electrode to repel the monomer adsorbed at the hot spot into the fluid lumen.

The invention also provides a biomolecule reading method, which comprises the following steps:

(1) the patch clamp system records the ionic current across the needle-tip nanopore and the blocking current signal of each cross-pore event;

(2) the laser Raman microscope emits laser to one side of the composite nanopore spectrum nanopore, and when the biomolecule monomer is located in a spectrum nanopore hot spot region, a Raman spectrum signal is generated, so that the composite nanopore spectrum nanopore has excellent discrimination and chemical sensitivity;

(3) the spectrum measuring device measures the Raman spectrum signal to obtain measurement data;

(4) the data acquisition and analysis device analyzes the measurement data, firstly judges the type of the basic nucleotide through the blocking current signal in the cross-pore event, records the position of the modified nucleotide in the single-stranded biomolecule through the time interval of the cross-pore event, then judges the type of the modified nucleotide through the spectral measurement data, and finally outputs the result.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Beijing institute of genomics (national center for bioinformatics)

<120> RNA exonuclease-nanopore complex and preparation method and application thereof

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Claims (10)

1. An exonuclease-nanopore complex comprising:

a nanopore structure;

an RNA exonuclease immobilized within the nanopore structure;

the RNA exonuclease has different speeds in hydrolyzing the basic nucleotide monomer and the modified nucleotide monomer.

2. The exonuclease-nanopore complex according to claim 1, wherein the nanopore structure is a protein nanopore; the protein nanopore performs fusion expression and self-assembly on the RNA exonuclease through a fusion protein technology to form a couplet of the RNA exonuclease and the protein nanopore.

3. The exorna-nanopore complex according to claim 2, wherein the protein nanopore is an α -hemolysin protein nanopore modifying β -cyclodextrin, MspA, CsgG or other nanopore useful for sequencing.

4. The exonuclease-nanopore complex according to claim 1, wherein the nanopore structure is a solid state nanopore; the solid-state nanopore leads and modifies RNA exonuclease near the solid-state nanopore by a click chemistry method to form a coupling structure of the RNA exonuclease and the solid-state nanopore.

5. The exonuclease-nanopore complex according to claim 1, wherein the exonuclease comprises an exonuclease from a prokaryote or a mutant thereof.

6. A method for preparing the RNA exonuclease-nanopore complex of any one of claims 1 to 5, comprising the steps of:

a) constructing a solid supporting structure of the RNA exonuclease nanopore complex,

b) embedding the RNA exonuclease-protein nanopore fusion protein in the solid support structure to form an RNA exonuclease-nanopore complex; or preparing a needle tip nanopore in a solid support structure, and then fixing the RNA exonuclease near the edge of the solid support structure to form an RNA exonuclease-nanopore complex.

7. A method of sequencing RNA comprising the steps of:

using the RNA exonuclease-nanopore complex as claimed in any one of claims 1 to 5, firstly using RNA exonuclease to sequentially cut off single nucleotides from one end of a sequencing sample and pass through a nanopore, and then using the characteristic that the nucleotides pass through the nanopore to judge basic nucleotides and modified nucleotides;

if the judgment result is the basic nucleotide, recording the type and the sequence of AUCG;

if the judgment result is the modified nucleotide, recording the position of the modified nucleotide, and detecting the chemically modified type of the modified nucleotide;

through the process, the sequence information of the nucleotide of the sequencing sample is obtained, and the direct reading of the RNA sequence is realized.

8. The sequencing method according to claim 7, wherein the process of determining the base nucleotide and the modified nucleotide by using the characteristic of the nucleotide passing through the nanopore specifically comprises the following steps:

high salt solution is poured into two sides of the nanopore, positive and negative electrodes are applied to form transmembrane voltage and ion current crossing the nanopore, and a patch clamp amplification circuit is applied to record the ion current;

nucleotide enters the high-salt solution from the negative electrode side, passes through the nanopore under the driving of an electric field and gushes to the positive electrode;

and recording the transient reduction of the ion current signal when the nucleotide passes through the nanopore as a one-time hole crossing event, and judging whether the nucleotide passing through the nanopore is a basic nucleotide or a modified nucleotide according to the time interval between the time when the nucleotide passes through the nanopore and the previous hole crossing event.

9. The sequencing method according to claim 8, wherein a Y-shaped nano flow channel is designed on one side of the nanopore positive electrode, and a basic nucleotide capture electrode and a modified nucleotide capture electrode are respectively arranged in two branched channels;

the basic nucleotide capture electrode is opened to drive the mononucleotide to pass through the nanopore and enter the channel where the mononucleotide is located; when the modified nucleotide passes through the nanopore, the time interval of the modified nucleotide is increased, the modified nucleotide capture electrode is started through the feedback circuit design, so that the capture voltage of the modified nucleotide capture electrode is higher than that of the basic nucleotide capture electrode, the modified nucleotide is adsorbed to another channel, and the separation of the modified nucleotide is realized;

after the modified nucleotide enters the branched channel through the nanopore, the capture voltage of the modified nucleotide is rapidly reduced to be lower than that of the basic nucleotide capture electrode, and the subsequent basic nucleotide is ensured to enter the correct channel.

10. The sequencing method of claim 7, wherein the method for detecting the chemically modified species of modified nucleotides is selected from one or more of single molecule Raman spectroscopy, fluorescence spectroscopy, infrared spectroscopy, absorption spectroscopy, reflection spectroscopy, mass spectroscopy, dielectric properties, and energy spectroscopy.

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