CN115029415B

CN115029415B - Preparation method and application of RNA vector related shift complex

Info

Publication number: CN115029415B
Application number: CN202210695244.0A
Authority: CN
Inventors: 樊军; 王佳佳
Original assignee: Chengdu Bomeda Technology Co ltd
Current assignee: Chengdu Bomeda Technology Co ltd
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2025-07-29
Anticipated expiration: 2042-06-17
Also published as: CN115029415A

Abstract

The application relates to the technical field of biomarkers, in particular to a preparation method and application of an RNA carrier related displacement complex, wherein the displacement complex comprises an RNA carrier, the RNA carrier comprises an RNA carrier core sequence, a biotin-marked primer and a digoxin-marked primer, the forward sequence of the RNA carrier core sequence is shown as SEQ ID NO.1, the reverse sequence of the RNA carrier core sequence is shown as SEQ ID NO.2, and one end of the RNA carrier is connected with a streptomycin-coated magnetic bead through the biotin-marked primer through the RNA carrier comprising the RNA carrier core sequence, the biotin-marked primer and the digoxin-marked primer, and the other end of the RNA carrier is connected to the surface of digoxin-resistant glass, so that the combination is firm. The RNA carrier core region can form a displacement complex with Holo-RdRp polymerase and nsp13 helicase, and the dynamic characteristics of nsp13 reaction can be effectively reflected in real time by using a single-molecule magnetic forceps technology.

Description

Preparation method and application of RNA vector related shift complex

Technical Field

The application relates to the technical field of markers, in particular to a preparation method and application of an RNA vector related shift complex.

Background

The replication of the RNA of the novel coronavirus genetic material is mediated by an RNA-dependent RNA polymerase (RdRp, encoded by non-structural protein 12, abbreviated as nsp 12), acting in Holo-RdRp polymerase (holo-RdRp, including nsp7/nsp8 ₂/nsp 12), synthesizing all viral RNA molecules, and thus RdRp is the core component of the viral non-structural protein (nsp) replication/transcription catalytic complex, although RdRp itself has only little activity, the addition of nsp7 and nsp8 cofactors enhances its template binding and processing capacity, thereby releasing the full capacity to replicate long-chain RNAs, which has been proposed as a target for nucleotide analogue antiviral drugs, such as the nucleotide analogue radcivir (REMDESIVIR), due to its important role in the RNA viral life cycle. While the novel coronavirus helicase (also known as nsp 13) is highly conserved among the novel coronaviruses, is an essential functional enzyme in viral replication and interacts with the host immune system.

Therefore, by researching the physiological activities of RdRp and nsp13 in the transmission process of the novel coronavirus, a theoretical basis can be provided for developing an inhibitor for blocking the novel coronavirus from entering cells. However, the traditional research on biological macromolecules such as proteins, DNA and the like is mostly performed on a systematic level by using biochemical methods, and along with the development of new technologies, a plurality of new research methods, such as optical tweezers, magnetic tweezers and atomic force microscopy, are developed, and simultaneously, a researched object can be observed on a smaller scale by combining a near-field microscope, for example, single-molecule magnetic tweezers once reveal a molecular mechanism of an anticancer drug Topotecan (Topotecan) for preventing topoisomerase I from unwinding, but currently, for the research on new coronaviruses, the dynamic characteristics of nsp13 reaction cannot be effectively revealed in real time if single-molecule magnetic tweezers are adopted, so that how to effectively reveal the dynamic characteristics of nsp13 reaction in real time is a technical problem which needs to be solved at present.

Disclosure of Invention

The application provides a preparation method and application of an RNA carrier related shift complex, which are used for solving the technical problem that the dynamic characteristics of nsp13 reaction in the prior art are difficult to effectively monitor in real time.

In a first aspect, the application provides an RNA vector, which comprises an RNA vector core sequence, a biotin-labeled primer and a digoxin-labeled primer, wherein the biotin-labeled primer is arranged at the 5 '-end of the RNA vector core sequence, the digoxin-labeled primer is arranged at the 3' -end of the RNA vector core sequence, the RNA vector core sequence comprises a forward sequence p-RNA and a reverse sequence t-RNA, the sequence of the forward sequence p-RNA is shown as SEQ ID NO.1, and the sequence of the reverse sequence t-RNA is shown as SEQ ID NO. 2.

Optionally, the RNA vector further comprises a transcription regulatory sequence, wherein the transcription regulatory sequence is arranged at the 5 'end of the RNA vector core sequence, and the biotin-labeled primer is arranged at the 5' end of the transcription regulatory sequence.

In a second aspect, the application provides a transcriptional extension complex comprising Holo-RdRp polymerase and the RNA vector of the first aspect, the subunits of the Holo-RdRp polymerase comprising the nsp12 subunit, the nsp7 subunit, and the nsp8 subunit.

In a third aspect, the application provides an RNA vector-associated translocation complex comprising nsp13 helicase and the transcriptional extension complex of the second aspect.

In a fourth aspect, the present application provides a method for preparing an RNA vector-associated shift complex, the method comprising:

constructing the RNA vector of the first aspect;

The gene segment of the nsp12 subunit in the second aspect and the gene segment of the nsp13 helicase in the third aspect are subjected to probe labeling to obtain a fluorescent probe labeled nsp12 subunit gene segment and a fluorescent probe labeled nsp13 helicase gene segment;

Carrying out plasmid transformation on the nsp12 subunit gene fragment marked by the fluorescent probe, and then carrying out expression and purification to obtain nsp12 subunit protein marked by the fluorescent probe;

carrying out biotin labeling on the nsp12 subunit protein containing the fluorescent probe label to obtain a double-labeled nsp12 subunit protein containing biotin label exchange and fluorescent probe label;

Purifying the nsp7 subunit and the nsp8 subunit of the second aspect respectively, and adding the double-labeled nsp12 subunit protein to mix to obtain Holo-RdRp polymerase containing the purified nsp7 subunit protein, the purified nsp8 subunit protein and the double-labeled nsp12 subunit protein;

Performing a first incubation reaction on the RNA vector and the Holo-RdRp polymerase to obtain a transcription extension complex;

carrying out plasmid transformation on the nsp13 helicase gene fragment marked by the fluorescent probe, and then carrying out expression and purification to obtain purified nsp13 helicase protein;

Mixing the transcription elongation complex and the purified nsp13 helicase protein in a preset volume, and then adding ADP-AlF3 for a second incubation reaction to obtain a translocation complex.

Optionally, the plasmid transformation is performed on the nsp12 subunit gene fragment marked by the fluorescent probe, and then the expression and the purification are performed, so that the nsp12 subunit protein containing the fluorescent probe mark is obtained, which specifically comprises the following steps:

fusing pRSFDuet-1 plasmid containing nsp12 subunit gene fragment marked by fluorescent probe and avi-tag plasmid to obtain compound plasmid;

converting the composite plasmid into competent cells of escherichia coli, and performing IPTG induction and lysis to obtain a lysate;

and collecting the cleavage product, and screening and filtering by a purification column to obtain the purified fluorescent probe marked nsp12 subunit protein.

Optionally, the step of carrying out biotin labeling on the nsp12 subunit protein containing the fluorescent probe label to obtain a double-labeled nsp12 subunit protein containing the biotin label exchange and the fluorescent probe label specifically comprises the following steps:

And carrying out a third incubation reaction on the purified fluorescent probe-labeled nsp12 subunit protein and biotin by using ligase to obtain double-labeled nsp12 subunit protein.

Optionally, the purification method of the nsp7 subunit or the nsp8 subunit comprises:

converting pCDFduet plasmid containing nsp7 subunit gene fragment or nsp8 subunit gene fragment into competent cells of escherichia coli, and performing IPTG induction and cleavage to obtain a cleavage product;

collecting the cleavage product, and screening and filtering by a purification column to obtain purified nsp7 subunit protein or purified nsp8 subunit protein.

Optionally, the plasmid transformation is performed on the nsp13 helicase gene fragment marked by the fluorescent probe, and then the expression and the purification are performed to obtain the purified nsp13 helicase protein, which specifically comprises the following steps:

Converting pet28 plasmid containing nsp13 subunit gene fragment marked by fluorescent probe into competent cells of escherichia coli, and performing IPTG induction and cleavage to obtain a cleavage product;

collecting the cleavage product, and screening and filtering by a purification column to obtain the purified nsp13 subunit protein.

In a fourth aspect, the application provides the use of an RNA vector-associated translocation complex, comprising using the translocation complex of the third aspect in single molecule magnetic tweezer techniques for the kinetic characterization of nsp13 reactions.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

according to the RNA carrier provided by the embodiment of the application, through the RNA carrier comprising the RNA carrier core sequence, the biotin-marked primer and the digoxin-marked primer are respectively designed at two ends of the RNA carrier core sequence, one end of the RNA carrier is connected with a streptomycin-wrapped magnetic bead through the biotin-marked primer, and the other end of the RNA carrier is connected with the anti-digoxin-treated glass surface, so that the RNA carrier is firmly combined, the fluctuation change of the extension of an RNA chain is reflected by measuring the distance between the magnetic bead and the glass surface, meanwhile, the set RNA carrier core can form a transcription extension complex together with Holo-RdRp polymerase, and the formed transcription extension complex is matched with nsp13 helicase to form a displacement complex, so that the dynamic characteristics of nsp13 reaction can be effectively monitored in real time in a single-molecule magnetic tweezers technology by observing the reaction and the process of the nsp13 helicase through a microscope.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a schematic diagram of the RNA vector core sequence of the RNA vector according to the embodiment of the present application;

FIG. 2 is a schematic diagram of the structure of an RNA vector according to an embodiment of the present application;

FIG. 3 is a schematic diagram showing the binding of RNA carrier to magnetic beads and glass slides according to an embodiment of the present application;

FIG. 4 is a schematic flow chart of a method according to an embodiment of the present application;

FIG. 5 is a detailed flow chart of a method according to an embodiment of the present application;

FIG. 6 is a continuation of FIG. 5;

FIG. 7 is a schematic diagram of the construction of a transcriptionally extended complex according to an embodiment of the present application;

FIG. 8 is a schematic diagram of the construction of a displaced complex provided by an embodiment of the present application;

FIG. 9 is a schematic diagram of a dynamic process for researching a displacement complex by using single-molecule magnetic tweezers and a microscope according to an embodiment of the application;

FIG. 10 is a schematic diagram of the composition of an optical path using a single-molecule magnetic tweezer and a microscope according to an embodiment of the present application;

FIG. 11 is a schematic diagram of a dynamic process for real-time tracking of whether the nsp13 and the transcriptional extension complex form a shift complex and their directions by using a single-molecule magnetic tweezer coupled fluorescence technique according to an embodiment of the present application;

FIG. 12 is a schematic diagram showing the dynamic process of tracking nsp13 helicase and transcriptionally extended complexes, and assembling to form displaced complexes, according to an embodiment of the present application.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

The inventive thinking of the application is:

the magnetic forceps can record the real-time position of the biomacromolecule connected with the magnetic beads, detect the extension change of the RNA chain, pull the magnetic beads out of the TIR evanescent field by applying external force by using the magnetic forceps, effectively remove the fluorescence interference of the magnetic beads, improve the signal-to-noise ratio and track the dynamic change of the biomolecules marked by the probes.

In the single-molecule magnetic tweezer technology, the real-time position of the biomacromolecule connected with the magnetic beads needs to be recorded in real time through the magnetic tweezer, so that the connection degree between the shift complex connected with the nsp13 and the magnetic beads needs to be ensured to be firm, further the physiological activity of the nsp13 in the new coronavirus can be accurately reflected and researched, and therefore, the shift complex needs to be designed in a correlation manner, so that the shift complex can be firmly combined with the magnetic beads, and the dynamic characteristics of the nsp13 reaction can be effectively revealed, but in the current single-molecule magnetic tweezer exploration process, the combination strength between the shift complex connected with the nsp13 and the magnetic beads or the connection degree of the shift complex nsp13 is not high, and the kinematic characteristics in the nsp13 reaction process can not be effectively reflected.

The technical scheme provided by the embodiment of the invention aims to solve the technical problems, and the overall thought is as follows:

in one embodiment of the application, an RNA vector is provided, the RNA vector comprises an RNA vector core sequence, a biotin-labeled primer and a digoxin-labeled primer, the biotin-labeled primer is arranged at the 5 '-end of the RNA vector core sequence, the digoxin-labeled primer is arranged at the 3' -end of the RNA vector core sequence, the RNA vector core sequence comprises a forward sequence p-RNA and a reverse sequence t-RNA, the sequence of the forward sequence p-RNA is shown as SEQ ID NO.1, and the sequence of the reverse sequence t-RNA is shown as SEQ ID NO. 2.

In some alternative embodiments, the RNA vector further comprises a transcription regulatory sequence disposed 5 'to the RNA vector core sequence, and the biotin-labeled primer is disposed 5' to the transcription regulatory sequence.

In the embodiment of the application, the control RNA vector also comprises TRS (transcription regulation sequence) sequences, which is helpful for researching the regulation and control of the TRS sequence in the nsp13 reaction transcription termination, so that the converter planting process of the nsp13 reaction process can be reflected.

In one embodiment of the present application, a transcription elongation complex is provided, comprising Holo-RdRp polymerase and the RNA vector, the subunits of the Holo-RdRp polymerase comprising the nsp12 subunit, the nsp7 subunit, and the nsp8 subunit.

In one embodiment of the application, an RNA vector-associated translocation complex is provided, the translocation complex comprising nsp13 helicase and the transcriptional extension complex.

In the embodiment of the application, the control displacement complex can comprise nsp13 helicase, so that the transcription extension complex formed by the RNA carrier is matched with the nsp13 helicase to form the displacement complex, and thus, in the single-molecule magnetic forceps technology, the reaction and the process participated by the nsp13 helicase can be observed through a microscope, and the dynamic characteristics of the nsp13 reaction can be effectively monitored in real time.

In one embodiment of the present application, as shown in FIG. 4, there is provided a method for preparing an RNA vector-associated shift complex, the method comprising:

S1, constructing the RNA vector;

S2, carrying out probe marking on the gene fragment of the nsp12 subunit and the gene fragment of the nsp13 helicase to obtain a fluorescent probe marked nsp12 subunit gene fragment and a fluorescent probe marked nsp13 helicase gene fragment;

s3, carrying out plasmid transformation on the nsp12 subunit gene fragment marked by the fluorescent probe, and then carrying out expression and purification to obtain nsp12 subunit protein containing the fluorescent probe mark;

s4, carrying out biotin labeling on the nsp12 subunit protein containing the fluorescent probe label to obtain double-labeled nsp12 subunit protein containing biotin label exchange and fluorescent probe label;

S5, respectively purifying the nsp7 subunit and the nsp8 subunit, adding double-labeled nsp12 subunit proteins, and mixing to obtain Holo-RdRp polymerase containing purified nsp7 subunit proteins, purified nsp8 subunit proteins and double-labeled nsp12 subunit proteins;

s6, performing a first incubation reaction on the RNA carrier and the Holo-RdRp polymerase to obtain a transcription extension complex;

s7, carrying out plasmid transformation on the nsp13 helicase gene fragment marked by the fluorescent probe, and then carrying out expression and purification to obtain purified nsp13 helicase protein;

S8, mixing the transcription extension complex and the purified nsp13 helicase protein in a preset volume, and then adding ADP-AlF3 for a second incubation reaction to obtain a translocation complex.

In some optional embodiments, the temperature of the first incubation reaction is 25 ℃ to 35 ℃, and the time of the first incubation reaction is 25min to 35min;

The temperature of the second incubation reaction is 25-35 ℃, and the time of the second incubation reaction is 2-8 min.

In the embodiment of the application, the temperature of the first incubation reaction is 25-35 ℃, and the positive effect is that the incubation reaction between the RNA carrier and the purified Holo-RdRp polymerase is complete within the temperature range, so that the purified nsp7 subunit protein, the purified nsp8 subunit protein and the biotin-marked nsp12 subunit protein can exist stably in the RNA carrier, and when the value of the temperature is larger or smaller than the end value of the range, the purified Holo-RdRp polymerase cannot be combined with the RNA carrier stably, and the formation of the final transcription extension complex is affected.

The first incubation reaction time is 25-35 min, and has the advantages that the incubation reaction between the RNA carrier and purified Holo-RdRp polymerase is complete within the time range, so that purified nsp7 subunit protein, purified nsp8 subunit protein and biotin-marked nsp12 subunit protein can exist in the RNA carrier stably, the time value is larger or smaller than the end value of the range, if the time value is too long, the reaction time is prolonged, and if the time value is too short, the purified Holo-RdRp polymerase cannot be combined with the RNA carrier stably, and the formation of a final transcription extension complex is affected.

The second incubation reaction temperature is 25-35 ℃, and has the advantages that the incubation reaction between the transcriptional elongation complex and the purified nsp13 helicase is complete in the temperature range, so that the nsp13 helicase stably exists in the transcriptional elongation complex, and when the temperature value is larger or smaller than the end value of the range, the purified nsp13 helicase cannot be stably combined with the RNA carrier in the transcriptional elongation complex, and the final formation of the shift complex is affected.

The second incubation reaction time is 2 min-8 min, and has the positive effects that in the time range, the incubation reaction between the transcriptional extension complex and the purified nsp13 helicase is complete, so that the nsp13 helicase stably exists in the transcriptional extension complex, the time value is larger or smaller than the end value of the range, if the time value is too long, the reaction time is prolonged, and if the time value is too short, the purified nsp13 helicase cannot be stably combined with the transcriptional extension complex, and the molding of the final transcriptional extension complex is affected.

In some alternative embodiments, the predetermined volume is 0.5-1.5:2.

In the embodiment of the application, the positive effect of controlling the preset volume to be 0.5-1.5:2 is that the transcription elongation complex and nsp13 protein are fully mixed, so that the formation of a displacement complex is sufficient.

In some alternative embodiments, as shown in fig. 5, the plasmid transformation is performed on the nsp12 subunit gene fragment labeled by the fluorescent probe, and then the expression and purification are performed, so as to obtain a nsp12 subunit protein containing the fluorescent probe label, which specifically includes:

S31, fusing pRSFDuet-1 plasmid containing a fluorescent probe marked nsp12 subunit gene fragment and avi-tag plasmid to obtain a composite plasmid;

S32, converting the composite plasmid into competent cells of escherichia coli, and performing IPTG induction and lysis to obtain a lysate;

s33, collecting the cleavage product, and screening and filtering by a purification column to obtain the purified fluorescent probe marked nsp12 subunit protein.

In the embodiment of the application, the expression of the nsp12 subunit is enhanced by adopting a composite plasmid form, so that the nsp12 subunit protein marked by a sufficient fluorescent probe can be obtained.

In some alternative embodiments, the biotin-labeling of the nsp12 subunit protein comprising fluorescent probe labeling results in a dual-labeled nsp12 subunit protein comprising a biotin-labeled shift and fluorescent probe labeling, specifically comprising:

S41, carrying out a third incubation reaction on the purified fluorescent probe-labeled nsp12 subunit protein and biotin by using ligase to obtain the double-labeled nsp12 subunit protein containing biotin label exchange and fluorescent probe label.

In the embodiment of the application, the fluorescent probe marked nsp12 subunit protein is marked by biotin, so that the magnetic beads can be firmly combined with the nsp12 subunit protein, thereby avoiding the falling of Holo-RdRp polymerase and effectively eliminating the interference matters affecting the nsp13 reaction process.

In some alternative embodiments, the method of purifying the nsp7 subunit or the nsp8 subunit comprises:

In some alternative embodiments, as shown in fig. 6, the plasmid transformation of the fluorescent probe-labeled nsp13 helicase gene fragment, followed by expression and purification, yields a purified nsp13 helicase protein, specifically comprising:

S71, converting pet28 plasmid containing a fluorescent probe marked nsp13 subunit gene fragment into competent cells of escherichia coli, and performing IPTG induction and cleavage to obtain a cleavage product;

s72, collecting the cleavage product, and screening and filtering by a purification column to obtain the purified nsp13 subunit protein.

In the embodiment of the application, the nsp13 subunit protein can be further purified by transforming the plasmid of the nsp13 subunit gene fragment marked by the fluorescent probe into competent cells and then carrying out induction and cleavage.

In one embodiment of the application, there is provided the use of an RNA vector-associated translocation complex, comprising using the translocation complex in single molecule magnetic tweezer techniques for the kinetic characterization of nsp13 reactions.

Example 1

S1, constructing an RNA vector, which specifically comprises the following steps:

Because SARS-CoV-2 uses RNA as genetic material, and the reaction substrates of Holo-RdRp polymerase and nsp13 helicase are RNA, it is important to construct the core sequence of RNA carrier, and the construction method of the core sequence of RNA carrier is shown in figure 1, and the core sequence of RNA carrier constructed by the application includes:

Forward sequence p-RNA 5'-CGCGUAGCAUGCUACGUCAUUCUCCUAAGAAGCUA-3', and reverse sequence t-RNA 3'-GCGCAUCGUACGAUGCAGUAAGAGGAUUCUUCGAU-5',

As shown in FIG. 2, a section of biotin-labeled 1kb random sequence is connected to the 5 'end of the core sequence as a linker fragment, a section of digoxin-labeled 1kb random sequence is connected to the 3' end of the core sequence as a linker fragment, and as shown in FIG. 3, the RNA carrier core sequence can be connected to a streptomycin-coated magnetic bead at one end and to the anti-digoxin-treated glass surface at the other end by such treatment, and further the fluctuation and variation of the RNA chain extension of the carrier can be reflected only by measuring the distance from the magnetic bead to the glass surface.

To further investigate the regulatory mechanism of TRS sequences for converter planting, the present application also inserts TRS sequences into RNA vectors, specifically at positions between the biotin tag and the RNA vector core sequence as shown in FIG. 2.

Example 2

Example 2 and example 1 were compared, and the difference between example 2 and example 1 is that:

Further constructs of transcription elongation complex (RTC), specifically including:

s2, carrying out probe marking on a gene fragment of the nsp12 subunit and a gene fragment of the nsp13 helicase to obtain a fluorescent probe marked nsp12 subunit gene fragment and a fluorescent probe marked nsp13 helicase gene fragment, wherein the method specifically comprises the following steps:

Connecting SNAP expression sequences to the 3' ends of the nsp12 subunit gene fragment and the nsp13 helicase gene fragment through linker sequences, and then carrying out expression analysis to determine that the SNAP tag protein has a common equivalent expression quantity;

s3, carrying out plasmid transformation on the nsp12 subunit gene fragment marked by the fluorescent probe, and then carrying out expression and purification to obtain the nsp12 subunit protein containing the fluorescent probe mark, wherein the method specifically comprises the following steps:

S32, converting the composite plasmid into competent cells of escherichia coli BL21, and performing IPTG induction and lysis to obtain a lysate;

s33, collecting a lysate, sequentially screening and filtering the lysate by using three purification columns of HITRAP HEPARIN (GE biosystems), HISTRAP HP X2 (GE biosystems) and Superdex 200Hiload (GE biosystems) after the collected cells are cracked and broken, so as to obtain purified fluorescent probe-marked nsp12 subunit protein;

In order to ensure successful marking of the nsp12 subunit gene fragment, the purified SNAP marked nsp12 protein is incubated with SNAP-JF549 (Bio-Techne company) at room temperature, the nsp12 marking rate is detected, and if the nsp12 marking rate meets the expectation, the obtained nsp12 protein is reserved, so that the Holo-RdRp polymerase marked by the JF549 fluorescent probe can be obtained;

S4, carrying out biotin labeling on the nsp12 subunit protein containing the fluorescent probe label to obtain double-labeled nsp12 subunit protein containing biotin label exchange and fluorescent probe label, wherein the method specifically comprises the following steps:

S41, carrying out a third incubation reaction on the purified fluorescent probe marked nsp12 subunit protein and biotin by using ligase BirA, wherein the reaction temperature is 30 ℃ and the reaction time is 30min, so as to obtain the double marked nsp12 subunit protein containing biotin marked exchange and fluorescent probe marked.

S5, as shown in FIG. 7, purifying the nsp7 subunit and the nsp8 subunit respectively, adding the double-labeled nsp12 subunit protein, and mixing to obtain Holo-RdRp polymerase containing the purified nsp7 subunit protein, the purified nsp8 subunit protein and the double-labeled nsp12 subunit protein, wherein the purification method of the nsp7 subunit and the nsp8 subunit comprises the following steps:

converting pCDFduet plasmid containing nsp7 subunit gene fragment or nsp8 subunit gene fragment into competent cells of escherichia coli Eco BL21 (DE 3), and performing IPTG induction and cleavage to obtain a cleavage product;

collecting the cleavage products, and sequentially screening and filtering by using HISTRAP HP column (GE biosystems) and Superdex 75Hiload 16/600 (GE biosystems) purification columns to obtain purified nsp7 subunit proteins or purified nsp8 subunit proteins;

S6, performing a first incubation reaction on the RNA carrier and Holo-RdRp polymerase to obtain a transcription extension complex.

Through the steps, holo-RdRp polymerase marked by JF549 fluorescent probe can be obtained.

Example 3

Example 3 and example 2 are compared, and the difference between example 3 and example 2 is that:

S7, carrying out plasmid transformation on the nsp13 helicase gene fragment marked by the fluorescent probe, and then carrying out expression and purification to obtain purified nsp13 helicase protein, wherein the method specifically comprises the following steps:

S71, converting pet28 plasmid containing a fluorescent probe marked nsp13 subunit gene fragment into competent cells of escherichia coli Eco Rosetta (DE 3), and performing IPTG induction and cleavage to obtain a cleavage product;

S72, collecting the cleavage products, and sequentially screening and filtering by using purifying columns HISTRAP HP and Superdex 200Hiload 16/600 (GE biosystems) to obtain purified nsp13 subunit proteins.

S8, as shown in FIG. 8, mixing the transcription elongation complex and the purified nsp13 helicase protein at a ratio of 1:2, and then adding ADP-AlF3 with a concentration of 1M for a second incubation reaction, wherein the reaction temperature is 30 ℃ and the reaction time is 5min, so as to obtain a shift complex.

Example 4

Example 4 and example 3 were compared, and the difference between example 4 and example 3 is that:

an application of an RNA carrier related shift complex, which comprises the following specific steps:

mixing the displacement compound and the magnetic beads wrapped by streptomycin at normal temperature, connecting the displacement compound with the magnetic beads through the action of biotin and streptomycin to form an RTC compound-magnetic bead mixed system, placing the mixed system in a single-molecule magnetic forceps reaction chamber, combining anti-digoxin at the tail end of RNA with digoxin on the surface of glass, connecting the tail end of the RNA chain mark in the displacement compound on the surface of glass, and suspending the tail end of the non-fixed RNA in the reaction solution due to the external pulling force exerted by a magnetic field as shown in figure 9.

As shown in fig. 10, the magnetic forceps microscope is used to record the position information of the magnetic beads, so as to detect the position of the shift complex in real time, and since nsp13 helicase is introduced into the system of the shift complex, whether the shift complex is formed under the interaction between nsp13 helicase and the transcription extension complex can be tracked in real time, if so, the helicase mechanism of nsp13 can be detected in vitro by further observation.

As shown in FIG. 11, holo-RdRp transcriptional extension complex was constructed, external force was applied to RNA of Holo-RdRp transcriptional extension complex using single molecule magnetic tweezers, the RNA secondary structure was regulated, the effect of the RNA secondary structure on nsp13 regulation process was studied, whether fluorescent labeled Holo-RdRp was always present in the displaced complex was observed, and after dissociation of the displaced complex, the forward direction of Holo-RdRp was observed.

As shown in fig. 12, using fluorescence-labeled nsp13 helicase, the process of unwinding and translocation of nsp13 on the RNA strand was observed, along with how nsp13 assembled to form a translocated complex, and the process of dynamic dissociation of the translocated complex.

In conclusion, the dynamic characteristics of the nsp13 reaction can be effectively explored through the translocation complex system constructed by the application.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

The RNA carrier provided by the embodiment of the application comprises an RNA carrier core sequence, a biotin-marked primer and a digoxin-marked primer, wherein the biotin-marked primer and the digoxin-marked primer are respectively designed at two ends of the RNA carrier core sequence, so that one end of the RNA carrier is connected with a streptomycin-wrapped magnetic bead through the biotin-marked primer, and the other end of the RNA carrier is connected with the anti-digoxin-treated glass surface, thereby reflecting the fluctuation change of the extension of an RNA chain by measuring the distance between the magnetic bead and the glass surface, simultaneously, the set RNA carrier core can form a transcription extension complex together with Holo-RdRp polymerized holoenzyme, and the formed transcription extension complex is matched with nsp13 helicase to form a displacement complex, so that the dynamic characteristics of nsp13 reaction can be monitored effectively in real time by observing the reaction and the process of the nsp13 helicase through a microscope in a single-molecule magnetic forceps technology.

(2) The transcription extension complex provided by the embodiment of the application can find an intermediate state formed by a shift complex by utilizing Holo-RdRp polymerase marked by fluorescent protein SNAP-JF549 with strong light stability, further find an interaction product and an action rule of related helicase and an RNA substrate, reconstruct the complete shift complex, and study the influence of an RNA structure under the regulation of external force on the intermediate state of nsp 13-Holo-RdRp.

(3) According to the displacement complex provided by the embodiment of the application, through adding Holo-RdRp polymerase and nsp13 helicase marked by the fluorescent probe into a single-molecule detection system, the processes of assembling, displacing, dissociating and the like of the helicase displacement complex can be positioned and tracked in real time in a total internal reflection TIR evanescent field.

(4) The method provided by the embodiment of the application can be based on nsp13 helicase protein by assembling a shift complex containing fluorescent protein SNAP-JF549 label with strong light stability, can reveal the dynamic characteristics of nsp13 reaction on a single molecular level, provides a theoretical basis for researching a SARS-CoV-2 transcription regulation mechanism, provides a basis for searching an inhibitor for blocking viruses from entering cells, and also provides a basic thought for researching and developing antiviral agents targeting nsp13, holo-RdRp and the like.

It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising" does not exclude the presence of additional identical elements in a process, method, article, or apparatus that comprises an element.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the 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> Chengdu Bomaida technologies Co., ltd

<120> Preparation method and application of RNA vector related translocation complex

<140> CN202210695244.0

<141> 2022-06-17

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35

<212> DNA/RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

cgcguagcau gcuacgucau ucuccuaaga agcua 35

<210> 2

<211> 35

<212> DNA/RNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

gcgcaucgua cgaugcagua agaggauucu ucgau 35

Claims

1. An RNA vector-associated translocation complex, wherein the translocation complex comprises a nsp13 helicase and a transcriptional extension complex, the transcriptional extension complex comprising Holo-RdRp polymerase and an RNA vector;

The RNA carrier comprises an RNA carrier core sequence, a biotin-marked primer and a digoxin-marked primer, wherein the biotin-marked primer is arranged at the 5 'end of the RNA carrier core sequence, the digoxin-marked primer is arranged at the 3' end of the RNA carrier core sequence, the RNA carrier core sequence comprises a forward sequence p-RNA and a reverse sequence t-RNA, the sequence of the forward sequence p-RNA is shown as SEQ ID NO.1, and the sequence of the reverse sequence t-RNA is shown as SEQ ID NO. 2;

the RNA vector also comprises a TRS sequence, wherein the TRS sequence is arranged at the 5 'end of the core sequence of the RNA vector, and the biotin-labeled primer is arranged at the 5' end of the TRS sequence;

The subunits of Holo-RdRp polymerase include the nsp12 subunit, the nsp7 subunit, and the nsp8 subunit.

2. A method of preparing an RNA vector-associated shift complex, the method comprising:

constructing the RNA vector of claim 1;

carrying out probe marking on the gene fragment of the nsp12 subunit of claim 1 and the gene fragment of the nsp13 helicase of claim 1 to obtain a fluorescent probe marked nsp12 subunit gene fragment and a fluorescent probe marked nsp13 helicase gene fragment;

Purifying the nsp7 subunit and the nsp8 subunit of claim 1, respectively, and adding the double-labeled nsp12 subunit protein to mix to obtain Holo-RdRp polymerase containing the purified nsp7 subunit protein, the purified nsp8 subunit protein and the double-labeled nsp12 subunit protein;

3. The method according to claim 2, wherein said plasmid transformation of said fluorescent probe-labeled nsp12 subunit gene fragment, followed by expression and purification, yields a nsp12 subunit protein containing fluorescent probe labeling, specifically comprises:

4. The method according to claim 3, wherein said biotinylating said nsp12 subunit protein containing fluorescent probe label to obtain a double-labeled nsp12 subunit protein containing biotin label exchange and fluorescent probe label, specifically comprising:

5. The method according to claim 2, wherein the purification method of the nsp7 subunit or the nsp8 subunit comprises:

6. The method according to claim 2, wherein the plasmid transformation of the fluorescent probe-labeled nsp13 helicase gene fragment followed by expression and purification yields a purified nsp13 helicase protein, specifically comprising: