Preparation method and application of an RNA carrier-related translocation complex

Abstract

This application relates to the technical field of biomarkers, and particularly to a preparation method and application of an RNA vector-related translocation complex; the translocation complex includes an RNA vector, and the RNA vector includes an RNA vector core sequence, a biotin-labeled primer, and a digoxigenin-labeled primer. The forward sequence of the RNA vector core sequence is shown in SEQ ID NO.1, and the reverse sequence of the RNA vector core sequence is shown in SEQ ID NO.2; through the RNA vector including the RNA vector core sequence, the biotin-labeled primer, and the digoxigenin-labeled primer, one end of the RNA vector is connected to streptomycin-coated magnetic beads through the biotin-labeled primer, and the other end is connected to the digoxigenin-treated glass surface, with firm binding. The RNA vector core region can form a translocation complex with the Holo-RdRp polymerase and the nsp13 helicase, and the single-molecule magnetic tweezer technology can effectively reflect the kinetic characteristics of the nsp13 reaction in real time.

Description

Technical Field

[0001] This application relates to the technical field of markers, and particularly to a preparation method and application of an RNA vector-related translocation complex. Background Art

[0002] The replication of the genetic material RNA of the novel coronavirus is mediated by the RNA-dependent RNA polymerase (RdRp, encoded by non-structural protein 12, abbreviated as nsp12), and functions in the Holo-RdRp polymerase holoenzyme (holo-RdRp, including nsp7 / nsp82 / nsp12) to synthesize all viral RNA molecules. Therefore, RdRp is the core component of the viral non-structural protein (nsp) replication / transcription catalytic complex. Although RdRp itself has only very little activity, the addition of nsp7 and nsp8 cofactors enhances its template binding and processing ability, thus releasing its full ability to replicate long-chain RNA. Due to its important role in the RNA virus life cycle, RdRp has been proposed as a target for nucleotide analog antiviral drugs, such as the nucleotide analog Remdesivir. At the same time, the novel coronavirus helicase (also known as nsp13) is highly conserved in the novel coronavirus, is an essential functional enzyme in virus replication, and interacts with the host immune system.

[0003] Therefore, by studying the physiological activities of RdRp and nsp13 during the transmission process of the novel coronavirus, it can provide a theoretical basis for the development of inhibitors that block the entry of the novel coronavirus into cells. However, traditional studies of biological macromolecules, such as proteins, DNA, etc., mostly use biochemical methods and are carried out at the system level. With the development of new technologies, many new research methods have emerged, such as optical tweezers, magnetic tweezers, atomic force microscopy technology, and combined with near-field microscopy, the objects under study can be observed at a smaller scale. For example, single-molecule magnetic tweezers have once revealed the molecular mechanism by which the anticancer drug Topotecan hinders the unwinding of topoisomerase I. However, for the current research on the novel coronavirus, if single-molecule magnetic tweezers are used, the kinetic characteristics of the nsp13 reaction cannot be effectively revealed in real time. Therefore, how to effectively reveal the kinetic characteristics of the nsp13 reaction in real time is a technical problem that urgently needs to be solved at present. Summary of the Invention

[0004] This application provides a preparation method and application of an RNA vector-related translocation complex to solve the technical problem that the kinetic characteristics of the nsp13 reaction in the prior art are difficult to monitor effectively in real time.

[0005] In a first aspect, the present application provides an RNA vector, which includes an RNA vector core sequence, a biotin-labeled primer, and a digoxin-labeled primer. The biotin-labeled primer is located at the 5' end of the RNA vector core sequence, and the digoxin-labeled primer is located at the 3' end of the RNA vector core sequence. The RNA vector core sequence includes a forward sequence p-RNA and a reverse sequence t-RNA. The sequence of the forward sequence p-RNA is as shown in SEQ ID NO.1, and the sequence of the reverse sequence t-RNA is as shown in SEQ ID NO.2.

[0006] Optionally, the RNA vector further includes a transcriptional regulatory sequence, which is located at the 5' end of the RNA vector core sequence, and the biotin-labeled primer is located at the 5' end of the transcriptional regulatory sequence.

[0007] In a second aspect, the present application provides a transcriptional elongation complex, which includes Holo-RdRp polymerase holoenzyme and the RNA vector described in the first aspect. The subunits of the Holo-RdRp polymerase holoenzyme include nsp12 subunit, nsp7 subunit, and nsp8 subunit.

[0008] In a third aspect, the present application provides an RNA vector-related translocation complex, which includes nsp13 helicase and the transcriptional elongation complex described in the second aspect.

[0009] In a fourth aspect, the present application provides a method for preparing an RNA vector-related translocation complex, which includes:

[0010] Constructing the RNA vector described in the first aspect;

[0011] Probe-labeling the gene fragments of the nsp12 subunit described in the second aspect and the gene fragments of the nsp13 helicase described in the third aspect to obtain fluorescent probe-labeled nsp12 subunit gene fragments and fluorescent probe-labeled nsp13 helicase gene fragments;

[0012] Performing plasmid transformation on the fluorescent probe-labeled nsp12 subunit gene fragments, followed by expression and purification to obtain nsp12 subunit proteins containing fluorescent probe labels;

[0013] Performing biotin labeling on the nsp12 subunit proteins containing fluorescent probe labels to obtain double-labeled nsp12 subunit proteins containing biotin labels and fluorescent probe labels;

[0014] Purify the nsp7 subunit and the nsp8 subunit described in the second aspect respectively, and then add the double-labeled nsp12 subunit protein for mixing to obtain the Holo-RdRp polymerase holoenzyme containing the purified nsp7 subunit protein, the purified nsp8 subunit protein, and the double-labeled nsp12 subunit protein;

[0015] Perform a first incubation reaction on the RNA vector and the Holo-RdRp polymerase holoenzyme to obtain a transcription elongation complex;

[0016] Transform the plasmid of the nsp13 helicase gene fragment labeled with a fluorescent probe, and then perform expression and purification to obtain the purified nsp13 helicase protein;

[0017] Mix the transcription elongation complex and the purified nsp13 helicase protein in a preset volume, and then add ADP-AlF3 for a second incubation reaction to obtain a translocation complex.

[0018] Optionally, the transformation of the plasmid of the nsp12 subunit gene fragment labeled with a fluorescent probe, and then performing expression and purification to obtain the nsp12 subunit protein containing the fluorescent probe label specifically includes:

[0019] Fuse the pRSFDuet-1 plasmid containing the nsp12 subunit gene fragment labeled with a fluorescent probe and the avi-tag plasmid to obtain a composite plasmid;

[0020] Transform the composite plasmid into the competent cells of Escherichia coli, and then perform IPTG induction and lysis to obtain a lysate;

[0021] Collect the lysate, and then perform screening and filtration with a purification column to obtain the purified nsp12 subunit protein labeled with a fluorescent probe.

[0022] Optionally, the biotinylation of the nsp12 subunit protein containing the fluorescent probe label to obtain the double-labeled nsp12 subunit protein containing the biotin label and the fluorescent probe label specifically includes:

[0023] Perform a third incubation reaction on the purified nsp12 subunit protein labeled with a fluorescent probe and biotin with a ligase to obtain the double-labeled nsp12 subunit protein.

[0024] Optionally, the purification method of the nsp7 subunit or the nsp8 subunit includes:

[0025] Transform the pCDFduet plasmid containing the nsp7 subunit gene fragment or the nsp8 subunit gene fragment into the competent cells of Escherichia coli, and then perform IPTG induction and lysis to obtain a lysate;

[0026] Collect the pyrolysis products, and then perform screening and filtration with a purification column to obtain purified nsp7 subunit protein or purified nsp8 subunit protein.

[0027] Optionally, transform the plasmid of the nsp13 helicase gene fragment labeled with a fluorescent probe, and then perform expression and purification to obtain purified nsp13 helicase protein, which specifically includes:

[0028] Transform the pet28 plasmid containing the nsp13 subunit gene fragment labeled with a fluorescent probe into the competent cells of Escherichia coli, and then perform IPTG induction and lysis to obtain pyrolysis products;

[0029] Collect the pyrolysis products, and then perform screening and filtration with a purification column to obtain purified nsp13 subunit protein.

[0030] In a fourth aspect, the present application provides an application of an RNA carrier-related translocation complex, and the application includes: using the translocation complex described in the third aspect in a single-molecule magnetic tweezer technology for studying the kinetic characteristics of the reaction of nsp13.

[0031] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0032] An RNA carrier provided by an embodiment of the present application, through an RNA carrier including an RNA carrier core sequence, a biotin-labeled primer, and a digoxin-labeled primer, using the biotin-labeled primer and the digoxin-labeled primer are respectively designed at both ends of the RNA carrier core sequence, connecting one end of the RNA carrier to a streptomycin-coated magnetic bead through the biotin-labeled primer, and connecting the other end of the RNA carrier to the surface of a glass treated with anti-digoxin, making the RNA carrier firmly bound, so as to reflect the fluctuation change of RNA strand extension by measuring the distance from the magnetic bead to the glass surface. At the same time, since the set RNA carrier core can form a transcription elongation complex with the Holo-RdRp polymerase, and the formed transcription elongation complex cooperates with the nsp13 helicase to form a translocation complex, in the single-molecule magnetic tweezer technology, the reaction and process participated by the nsp13 helicase can be observed through a microscope, so as to be able to monitor the kinetic characteristics of the nsp13 reaction in real time and effectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings here are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention.

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 Schematic diagram of the RNA vector core sequence of the RNA vector provided in the embodiment of the present application;

[0036] Figure 2 Schematic diagram of the structure of the RNA vector provided in the embodiment of the present application;

[0037] Figure 3 Schematic diagram of the combination of the RNA vector provided in the embodiment of the present application with magnetic beads and a glass slide;

[0038] Figure 4 Schematic diagram of the process flow of the method provided in the embodiment of the present application;

[0039] Figure 5 Schematic diagram of the detailed process flow of the method provided in the embodiment of the present application;

[0040] Figure 6 For Figure 5 continuation;

[0041] Figure 7 Schematic diagram of the construction of the transcription elongation complex provided in the embodiment of the present application;

[0042] Figure 8 Schematic diagram of the construction of the translocation complex provided in the embodiment of the present application;

[0043] Figure 9 Schematic diagram of the dynamic process of studying the translocation complex using single-molecule magnetic tweezers and a microscope provided in the embodiment of the present application;

[0044] Figure 10 Schematic diagram of the optical path composition of using single-molecule magnetic tweezers and a microscope provided in the embodiment of the present application;

[0045] Figure 11 Schematic diagram of the dynamic process of using single-molecule magnetic tweezer coupled fluorescence technology to track in real time whether nsp13 and the transcription elongation complex form a translocation complex and its whereabouts provided in the embodiment of the present application;

[0046] Figure 12 Schematic diagram of the dynamic process of tracking the helicase nsp13 and the transcription elongation complex to assemble into a translocation complex provided in the embodiment of the present application. Detailed implementation manners

[0047] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention rather than limit the present invention.

[0048] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0049] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0050] The creative thinking of this application is as follows:

[0051] Magnetic tweezers can record the real-time position of biomolecules connected to magnetic beads, detect the extension change of RNA strands, use the external force applied by magnetic tweezers to pull the magnetic beads out of the TIR evanescent field, effectively remove the fluorescence interference of the magnetic beads, improve the signal-to-noise ratio, and track the dynamic changes of biomolecules labeled with probes.

[0052] In single-molecule magnetic tweezer technology, since it is necessary to record the real-time position of biomolecules connected to magnetic beads through magnetic tweezers in real time, it is necessary to ensure a strong connection between the translocation complex connected with nsp13 and the magnetic beads, so as to accurately reflect the physiological activities of nsp13 in the new coronavirus. Therefore, it is necessary to carry out relevant designs on the translocation complex so that it can bind firmly to the magnetic beads, thereby effectively revealing the kinetic characteristics of the nsp13 reaction. However, in the current single-molecule magnetic tweezer exploration process, the binding strength between the translocation complex connected with nsp13 and the magnetic beads or the connection degree of the translocation complex nsp13 is not high, and the kinematic characteristics during the nsp13 reaction cannot be effectively reflected.

[0053] The technical solution provided by the embodiments of the present invention to solve the above technical problems has the following general idea:

[0054] In one embodiment of the present application, an RNA vector is provided. The RNA vector includes an RNA vector core sequence, a biotin-labeled primer, and a digoxin-labeled primer. The biotin-labeled primer is provided at the 5' end of the RNA vector core sequence, and the digoxin-labeled primer is provided at the 3' end of the RNA vector core sequence. The RNA vector core sequence includes a forward sequence p-RNA and a reverse sequence t-RNA. The sequence of the forward sequence p-RNA is as shown in SEQ ID NO.1, and the sequence of the reverse sequence t-RNA is as shown in SEQ ID NO.2.

[0055] In some alternative embodiments, the RNA vector further comprises a transcriptional regulatory sequence, which is disposed at the 5'-end of the core sequence of the RNA vector, and the biotin-labeled primer is disposed at the 5'-end of the transcriptional regulatory sequence.

[0056] In the embodiments of the present application, the control RNA vector further comprises a sequence of TRS (transcription regulation sequence), which helps to study the regulation of the transcriptional termination of the regulatory sequence TRS in the nsp13 reaction, so as to reflect the transcriptional process of the nsp13 reaction.

[0057] In an embodiment of the present application, a transcriptional elongation complex is provided, which comprises a Holo-RdRp polymerase and the RNA vector, and the subunits of the Holo-RdRp polymerase include an nsp12 subunit, an nsp7 subunit, and an nsp8 subunit.

[0058] In an embodiment of the present application, an RNA vector-related translocation complex is provided, which comprises an nsp13 helicase and the transcriptional elongation complex.

[0059] In the embodiments of the present application, the control translocation complex can include an nsp13 helicase, so that the transcriptional elongation complex formed by the RNA vector and the nsp13 helicase cooperate to form a translocation complex, so that in the single-molecule magnetic tweezer technology, the reaction and process participated by the nsp13 helicase can be observed through a microscope, and thus the kinetic characteristics of the nsp13 reaction can be monitored in real time and effectively.

[0060] In an embodiment of the present application, as Figure 4 shown, a method for preparing an RNA vector-related translocation complex is provided, and the method comprises:

[0061] S1. Construct the RNA vector;

[0062] S2. Probe-label the gene fragments of the nsp12 subunit and the nsp13 helicase to obtain a fluorescent probe-labeled nsp12 subunit gene fragment and a fluorescent probe-labeled nsp13 helicase gene fragment;

[0063] S3. Perform plasmid transformation on the fluorescent probe-labeled nsp12 subunit gene fragment, and then perform expression and purification to obtain a fluorescent probe-labeled nsp12 subunit protein;

[0064] S4. Biotinylate the nsp12 subunit protein labeled with a fluorescent probe to obtain a doubly labeled nsp12 subunit protein containing biotinylation and fluorescent probe labeling;

[0065] S5. Purify the nsp7 subunit and the nsp8 subunit respectively, and then add the doubly labeled nsp12 subunit protein for mixing to obtain a Holo-RdRp polymerase holoenzyme containing the purified nsp7 subunit protein, the purified nsp8 subunit protein, and the doubly labeled nsp12 subunit protein;

[0066] S6. Perform a first incubation reaction on the RNA vector and the Holo-RdRp polymerase holoenzyme to obtain a transcription elongation complex;

[0067] S7. Transform the plasmid with the fluorescent probe-labeled nsp13 helicase gene fragment, and then perform expression and purification to obtain the purified nsp13 helicase protein;

[0068] S8. Mix the transcription elongation complex and the purified nsp13 helicase protein in a preset volume, and then add ADP-AlF3 for a second incubation reaction to obtain a translocation complex.

[0069] In some alternative embodiments, the temperature of the first incubation reaction is 25°C to 35°C, and the time of the first incubation reaction is 25 min to 35 min;

[0070] The temperature of the second incubation reaction is 25°C to 35°C, and the time of the second incubation reaction is 2 min to 8 min.

[0071] In the embodiments of the present application, the positive effect of the temperature of the first incubation reaction being 25°C to 35°C is that within this temperature range, the incubation reaction between the RNA vector and the purified Holo-RdRp polymerase holoenzyme is complete, enabling the purified nsp7 subunit protein, the purified nsp8 subunit protein, and the biotinylated nsp12 subunit protein to stably exist in the RNA vector; when the temperature value is greater than or less than the endpoint values of this range, it will cause the purified Holo-RdRp polymerase holoenzyme to be unable to stably bind to the RNA vector, affecting the formation of the final transcription elongation complex.

[0072] The positive effect of the first incubation reaction for 25 min to 35 min is that within this time range, the incubation reaction between the RNA vector and the purified Holo-RdRp polymerase is complete, enabling the purified nsp7 subunit protein, the purified nsp8 subunit protein, and the biotin-labeled nsp12 subunit protein to stably exist in the RNA vector; when the value of the time is greater than or less than the endpoint values of this range, if the value of the time is too long, the reaction time will be prolonged, and if the value of the time is too short, the purified Holo-RdRp polymerase will not be able to stably bind to the RNA vector, affecting the formation of the final transcription elongation complex.

[0073] The positive effect of the second incubation reaction at 25 °C to 35 °C is that within this temperature range, the incubation reaction between the transcription elongation complex and the purified nsp13 helicase is complete, enabling the nsp13 helicase to stably exist in the transcription elongation complex; when the value of the temperature is greater than or less than the endpoint values of this range, the purified nsp13 helicase will not be able to stably bind to the RNA vector in the transcription elongation complex, affecting the formation of the final translocation complex.

[0074] The positive effect of the second incubation reaction for 2 min to 8 min is that within this time range, the incubation reaction between the transcription elongation complex and the purified nsp13 helicase is complete, enabling the nsp13 helicase to stably exist in the transcription elongation complex; when the value of the time is greater than or less than the endpoint values of this range, if the value of the time is too long, the reaction time will be prolonged, and if the value of the time is too short, the purified nsp13 helicase will not be able to stably bind to the transcription elongation complex, affecting the formation of the final transcription elongation complex.

[0075] In some alternative embodiments, the preset volume is 0.5 to 1.5:2.

[0076] In the embodiments of the present application, the positive effect of controlling the preset volume to be 0.5 to 1.5:2 is that the transcription elongation complex and the nsp13 protein are mixed sufficiently, enabling the formation of a sufficient translocation complex.

[0077] In some alternative embodiments, as Figure 5 shown, plasmid transformation of the nsp12 subunit gene fragment labeled with a fluorescent probe is performed, followed by expression and purification to obtain the nsp12 subunit protein labeled with a fluorescent probe, which specifically includes:

[0078] S31. Fusing the pRSFDuet-1 plasmid containing the nsp12 subunit gene fragment labeled with a fluorescent probe and the avi-tag plasmid to obtain a composite plasmid;

[0079] S32. Transform the composite plasmid into competent cells of Escherichia coli, followed by IPTG induction and lysis to obtain a lysate.

[0080] S33. Collect the lysate and then perform screening and filtration with a purification column to obtain purified nsp12 subunit protein labeled with a fluorescent probe.

[0081] In the embodiments of the present application, by adopting the form of a composite plasmid, the expression of the nsp12 subunit is enhanced, so that sufficient nsp12 subunit protein labeled with a fluorescent probe can be obtained.

[0082] In some alternative embodiments, biotin-label the nsp12 subunit protein labeled with a fluorescent probe to obtain a doubly labeled nsp12 subunit protein containing biotin labeling and fluorescent probe labeling, which specifically includes:

[0083] S41. Perform a third incubation reaction on the purified nsp12 subunit protein labeled with a fluorescent probe and biotin with a ligase to obtain a doubly labeled nsp12 subunit protein containing biotin labeling and fluorescent probe labeling.

[0084] In the embodiments of the present application, by labeling the nsp12 subunit protein labeled with a fluorescent probe with biotin, the magnetic beads can bind firmly to the nsp12 subunit protein, thereby avoiding the shedding of the Holo-RdRp polymerase holoenzyme and effectively removing the interfering substances that affect the nsp13 reaction process.

[0085] In some alternative embodiments, the purification method of the nsp7 subunit or the nsp8 subunit includes:

[0086] Transform the pCDFduet plasmid containing the nsp7 subunit gene fragment or the nsp8 subunit gene fragment into competent cells of Escherichia coli, followed by IPTG induction and lysis to obtain a lysate.

[0087] Collect the lysate and then perform screening and filtration with a purification column to obtain purified nsp7 subunit protein or purified nsp8 subunit protein.

[0088] In some alternative embodiments, as Figure 6 shown, the plasmid transformation of the nsp13 helicase gene fragment labeled with a fluorescent probe is followed by expression and purification to obtain purified nsp13 helicase protein, which specifically includes:

[0089] S71. Transform the pet28 plasmid containing the nsp13 subunit gene fragment labeled with a fluorescent probe into competent cells of Escherichia coli, followed by IPTG induction and lysis to obtain a lysate.

[0090] S72. Collect the pyrolysis products, and then screen and filter them with a purification column to obtain the purified nsp13 subunit protein.

[0091] In the embodiments of the present application, by transforming the plasmid of the nsp13 subunit gene fragment labeled with a fluorescent probe into competent cells and then inducing and lysing, the nsp13 subunit protein can be further purified.

[0092] In one embodiment of the present application, there is provided an application of an RNA vector-related translocation complex, and the application includes: using the translocation complex in a single-molecule magnetic tweezers technique for studying the kinetic characteristics of the reaction with nsp13.

[0093] Example 1

[0094] S1. Construct an RNA vector, specifically including:

[0095] Since SARS-CoV-2 uses RNA as its genetic material and the reaction substrates of Holo-RdRp polymerase and nsp13 helicase are both RNA, it is very important to construct the core sequence of the RNA vector. The construction method of the core sequence of the RNA vector is as Figure 1 shown. The core sequence of the RNA vector constructed in the present application includes:

[0096] Forward sequence p-RNA: 5’-CGCGUAGCAUGCUACGUCAUUCUCCUAAGAAGCUA-3’, and reverse sequence t-RNA: 3’-GCGCAUCGUACGAUGCAGUAAGAGGAUUCUUCGAU-5’,

[0097] As Figure 2 shown, a ~1 kb random sequence labeled with biotin is connected to the 5’ end of this core sequence as a linker fragment, and a ~1 kb random sequence labeled with digoxin is connected to its 3’ end as a linker fragment. As Figure 3 shown, by such treatment, one end of the core sequence of the RNA vector can be connected to streptavidin-coated magnetic beads, and the other end can be connected to the digoxin-treated glass surface. Furthermore, by simply measuring the distance from the magnetic beads to the glass surface, the fluctuation change of the RNA strand extension of the vector can be reflected.

[0098] In order to further study the regulation mechanism of the TRS sequence on converter planting, the present application also inserts the TRS sequence into the RNA vector, and the specific insertion position is as Figure 2 shown, between the biotin label and the core sequence of the RNA vector.

[0099] Example 2

[0100] Example 2 was compared with Example 1. The difference between Example 2 and Example 1 is as follows:

[0101] Further construct the transcription elongation complex (RTC), specifically including:

[0102] S2. Probe-label the gene fragment of the nsp12 subunit and the gene fragment of the nsp13 helicase to obtain the gene fragment of the nsp12 subunit labeled with a fluorescent probe and the gene fragment of the nsp13 helicase labeled with a fluorescent probe, specifically including:

[0103] Connect the SNAP expression sequence to the 3' ends of the gene fragment of the nsp12 subunit and the gene fragment of the nsp13 helicase through a linker sequence, and then perform expression analysis to determine that the SNAP-tag protein has an expression level comparable to that of the normal one;

[0104] S3. Perform plasmid transformation on the gene fragment of the nsp12 subunit labeled with a fluorescent probe, and then perform expression and purification to obtain the nsp12 subunit protein labeled with a fluorescent probe, specifically including:

[0105] S31. Fuse the pRSFDuet-1 plasmid containing the gene fragment of the nsp12 subunit labeled with a fluorescent probe and the avi-tag plasmid to obtain a composite plasmid;

[0106] S32. Transform the composite plasmid into the competent cells of Escherichia coli BL21, and then perform IPTG induction and lysis to obtain a lysate;

[0107] S33. Collect the lysate, and then screen and filter the collected cells through three purification columns, namely HiTrap Heparin (GE Biosciences), HisTrap HP X2 (GE Biosciences), and Superdex 200 Hiload (GE Biosciences) in sequence to obtain the purified nsp12 subunit protein labeled with a fluorescent probe;

[0108] To ensure the successful labeling of the gene fragment of the nsp12 subunit, the purified SNAP-labeled nsp12 protein is incubated with SNAP-JF549 (Bio-Techne) at room temperature to detect the labeling rate of nsp12. If it meets the expectation, the obtained nsp12 protein is retained, so that the Holo-RdRp polymerase labeled with the JF549 fluorescent probe can be obtained;

[0109] S4. Perform biotin labeling on the nsp12 subunit protein labeled with a fluorescent probe to obtain the doubly labeled nsp12 subunit protein containing biotin labeling and fluorescent probe labeling, specifically including:

[0110] S41. Perform the third incubation reaction on the purified fluorescent probe-labeled nsp12 subunit protein and biotin with ligase BirA at a reaction temperature of 30 °C for 30 min to obtain the nsp12 subunit protein with dual labeling of biotin labeling and fluorescent probe labeling.

[0111] S5. As Figure 7 shown, purify the nsp7 subunit and the nsp8 subunit respectively, and then add the dual-labeled nsp12 subunit protein for mixing to obtain the Holo-RdRp polymerase holoenzyme containing the purified nsp7 subunit protein, the purified nsp8 subunit protein, and the dual-labeled nsp12 subunit protein. Among them, the purification methods of the nsp7 subunit and the nsp8 subunit include:

[0112] Transform the pCDFduet plasmid containing the nsp7 subunit gene fragment or the nsp8 subunit gene fragment into the competent cells of Escherichia coli EcoBL21(DE3), and then perform IPTG induction and lysis to obtain the lysis product;

[0113] Collect the lysis product, and then sequentially perform screening filtration with the purification columns of HisTrap HP column (GE Biosciences) and Superdex 75 Hiload 16 / 600 (GE Biosciences) to obtain the purified nsp7 subunit protein or the purified nsp8 subunit protein;

[0114] S6. Perform the first incubation reaction on the RNA vector and the Holo-RdRp polymerase holoenzyme to obtain the transcription elongation complex.

[0115] After the above steps, the Holo-RdRp polymerase holoenzyme labeled with the JF549 fluorescent probe can be obtained.

[0116] Example 3

[0117] Compare Example 3 with Example 2. The differences between Example 3 and Example 2 are as follows:

[0118] S7. Perform plasmid transformation on the fluorescent probe-labeled nsp13 helicase gene fragment, and then perform expression and purification to obtain the purified nsp13 helicase protein, specifically including:

[0119] S71. Transform the pet28 plasmid containing the fluorescent probe-labeled nsp13 subunit gene fragment into the competent cells of Escherichia coli EcoRosetta(DE3), and then perform IPTG induction and lysis to obtain the lysis product;

[0120] S72. The lysate was collected and then filtered using HisTrap HP and Superdex 200 Hiload 16 / 600 (GE Bio) purification columns to obtain purified nsp13 subunit protein.

[0121] S8. Figure 8 As shown, the transcription elongation complex and the purified nsp13 helicase protein were mixed at a ratio of 1:2, and then 1M ADP-AlF3 was added for a second incubation reaction at a temperature of 30°C for 5 min to obtain a translocation complex.

[0122] Example 4

[0123] Comparing Example 4 with Example 3, the difference between Example 4 and Example 3 is:

[0124] An application of an RNA carrier-related translocation complex, comprising the following specific steps:

[0125] At room temperature, the translocation complex and streptomycin-coated magnetic beads are mixed together. The translocation complex is connected to the magnetic beads through the action of biotin and streptomycin to form an RTC complex-magnetic bead mixed system. The mixed system is then placed in a single-molecule magnetic tweezers reaction chamber. The anti-digoxigenin at the end of the RNA binds to the digoxigenin on the glass surface, and the labeled end of the RNA chain in the translocation complex is connected to the glass surface. Figure 9 As shown, the unimmobilized RNA ends are suspended in the reaction solution due to the exogenous pulling force applied by the magnetic field.

[0126] like Figure 10 As shown, by using a magnetic tweezers microscope, the position information of the magnetic beads can be recorded, and the position of the translocation complex can be detected in real time. Since the nsp13 helicase is introduced into the translocation complex system, it is possible to track in real time whether a translocation complex is formed under the interaction between the nsp13 helicase and the transcription elongation complex. If so, continued observation can be used to measure the unwinding mechanism of nsp13 in vitro.

[0127] like Figure 11 As shown, a Holo-RdRp transcription elongation complex was constructed, and single-molecule magnetic tweezers were used to apply external force to the RNA of the Holo-RdRp transcription elongation complex to regulate the RNA secondary structure. The influence of RNA secondary structure on the nsp13 regulatory process was studied, and whether the fluorescently labeled Holo-RdRp was always present in the translocation complex was observed. After the translocation complex dissociated, the whereabouts of the Holo-RdRp was observed.

[0128] like Figure 12As shown, by using the fluorescently labeled nsp13 helicase, the unwinding and translocation processes of nsp13 on the RNA strand were observed. At the same time, how nsp13 assembles to form a translocation complex and the dynamic dissociation process of the translocation complex were also observed.

[0129] In summary, through the translocation complex system constructed in this application, the kinetic characteristics of the nsp13 reaction can be effectively explored.

[0130] One or more technical solutions in the embodiments of this application at least have the following technical effects or advantages:

[0131] -(1) The RNA vector provided in the embodiment of this application, through the RNA vector including the RNA vector core sequence, biotin-labeled primer, and digoxin-labeled primer, with the biotin-labeled primer and digoxin-labeled primer designed at both ends of the RNA vector core sequence respectively, so that one end of the RNA vector is connected to the streptomycin-coated magnetic bead through the biotin-labeled primer, and the other end of the RNA vector is connected to the digoxin-treated glass surface. Thus, the fluctuation change of RNA strand extension is reflected by measuring the distance from the magnetic bead to the glass surface. At the same time, since the set RNA vector core can form a transcription elongation complex with the Holo-RdRp polymerase holoenzyme, and the formed transcription elongation complex cooperates with the nsp13 helicase to form a translocation complex, in the single-molecule magnetic tweezer technology, the reactions and processes participated by the nsp13 helicase can be observed through a microscope, so as to be able to monitor the kinetic characteristics of the nsp13 reaction in real time and effectively.

[0132] (2) The transcription elongation complex provided in the embodiment of this application can use the Holo-RdRp polymerase holoenzyme labeled with the highly photostable fluorescent protein SNAP-JF549 to discover the intermediate state formed by the translocation complex, and then discover the interaction products and interaction rules between the relevant helicase and the RNA substrate, reconstruct the complete translocation complex, and study the influence of the RNA structure under external force regulation on the nsp13-Holo-RdRp intermediate state.

[0133] (3) The translocation complex provided in the embodiment of this application, by adding the Holo-RdRp polymerase holoenzyme labeled with a fluorescent probe and the nsp13 helicase into the single-molecule detection system, in the total internal reflection TIR evanescent field, the processes of assembly, translocation, dissociation, etc. of the helicase translocation complex can be located and tracked in real time.

[0134] (4) The method provided by the embodiments of the present application can, by assembling a translocation complex labeled with the highly photo-stable fluorescent protein SNAP-JF549 and based on the nsp13 helicase protein, reveal the kinetic characteristics of the nsp13 reaction at the single-molecule level, provide a theoretical basis for studying the transcriptional regulation mechanism of SARS-CoV-2, and thus provide a basis for finding inhibitors that block virus entry into cells and also provide a basic idea for the development of antiviral drugs targeting nsp13, Holo-RdRp, etc.

[0135] It should be noted that in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, the elements defined by the statement "comprising..." do not exclude the presence of additional identical elements in the process, method, article or device comprising the elements.

[0136] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0137] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein. Sequence Listing <110> Chengdu Bomaida Technology Co., Ltd. <120> Preparation Method and Application of an RNA Vector-Related Translocation Complex <140> CN202210695244.0 <141> 2022-06-17 <160> 2 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 35 <212> DNA / RNA <213> Artificial Sequence <400> 1 cgcguagcau gcuacgucau ucuccuaaga agcua 35 <210> 2 <211> 35 <212> DNA / RNA <213> Artificial Sequence <400> 2 gcgcaucgua cgaugcagua agaggauucu ucgau 35

Claims

1. An RNA carrier-related translocation complex, characterized in that, The translocation complex includes nsp13 helicase and a transcription elongation complex, and the transcription elongation complex includes Holo-RdRp polymerase holoenzyme and an RNA carrier; The RNA carrier includes an RNA carrier core sequence, a biotin-labeled primer, and a digoxin-labeled primer. The biotin-labeled primer is located at the 5'-end of the RNA carrier core sequence, and the digoxin-labeled primer is located at the 3'-end of the RNA carrier core sequence. The RNA carrier core sequence includes 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 carrier further includes a TRS sequence, and the TRS sequence is located at the 5'-end of the RNA carrier core sequence. The biotin-labeled primer is located at the 5'-end of the TRS sequence; The subunits of the Holo-RdRp polymerase holoenzyme include an nsp12 subunit, an nsp7 subunit, and an nsp8 subunit.

2. A method for preparing an RNA vector-related translocation complex, characterized in that, The method includes: Constructing the RNA carrier according to claim 1; Probe-labeling the gene fragment of the nsp12 subunit according to claim 1 and the gene fragment of the nsp13 helicase according to claim 1 to obtain a fluorescent probe-labeled nsp12 subunit gene fragment and a fluorescent probe-labeled nsp13 helicase gene fragment; Performing plasmid transformation on the fluorescent probe-labeled nsp12 subunit gene fragment, followed by expression and purification to obtain a fluorescent probe-labeled nsp12 subunit protein; Performing biotin labeling on the fluorescent probe-labeled nsp12 subunit protein to obtain a dual-labeled nsp12 subunit protein containing biotin labeling and fluorescent probe labeling; Respectively purifying the nsp7 subunit and the nsp8 subunit according to claim 1, and then adding the dual-labeled nsp12 subunit protein for mixing to obtain a Holo-RdRp polymerase holoenzyme containing a purified nsp7 subunit protein, a purified nsp8 subunit protein, and a dual-labeled nsp12 subunit protein; Performing a first incubation reaction on the RNA carrier and the Holo-RdRp polymerase holoenzyme to obtain a transcription elongation complex; Performing plasmid transformation on the fluorescent probe-labeled nsp13 helicase gene fragment, followed by expression and purification to obtain a 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.

3. The method according to claim 2, wherein The performing plasmid transformation on the fluorescent probe-labeled nsp12 subunit gene fragment, followed by expression and purification to obtain a fluorescent probe-labeled nsp12 subunit protein specifically includes: Fusing a pRSFDuet-1 plasmid containing a fluorescent probe-labeled nsp12 subunit gene fragment and an avi-tag plasmid to obtain a composite plasmid; Transforming the composite plasmid into competent cells of Escherichia coli, followed by IPTG induction and lysis to obtain a lysate; Collect the pyrolysis products, and then screen and filter them with a purification column to obtain the purified fluorescent probe-labeled nsp12 subunit protein.

4. The method according to claim 3, wherein Biotin-label the nsp12 subunit protein containing the fluorescent probe label to obtain a double-labeled nsp12 subunit protein containing biotin label and fluorescent probe label, specifically including: Perform a third incubation reaction on the purified fluorescent probe-labeled nsp12 subunit protein and biotin with a ligase to obtain a double-labeled nsp12 subunit protein.

5. The method according to claim 2, wherein The purification method of the nsp7 subunit or the nsp8 subunit includes: Transform the pCDFduet plasmid containing the nsp7 subunit gene fragment or the nsp8 subunit gene fragment into the competent cells of Escherichia coli, and then perform IPTG induction and lysis to obtain pyrolysis products; Collect the pyrolysis products, and then screen and filter them with a purification column to obtain the purified nsp7 subunit protein or the purified nsp8 subunit protein.

6. The method according to claim 2, wherein Perform plasmid transformation on the fluorescent probe-labeled nsp13 helicase gene fragment, and then perform expression and purification to obtain the purified nsp13 helicase protein, specifically including: Transform the pet28 plasmid containing the fluorescent probe-labeled nsp13 subunit gene fragment into the competent cells of Escherichia coli, and then perform IPTG induction and lysis to obtain pyrolysis products; Collect the pyrolysis products, and then screen and filter them with a purification column to obtain the purified nsp13 subunit protein.

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