A single molecule method for detecting ribozyme isoforms

Abstract

The application aims to provide a method for detecting ribozyme isomers based on single molecule mechanics technology. The application first designs a DNA-RNA-DNA single molecule mechanics control structure object, and then actively detects the RNA ribozyme molecule through the single molecule technology, and measures the activity of the ribozyme molecule in the form of substrate combination. The method controls the folding state of the ribozyme by using the single molecule technology, analyzes the isomers of the ribozyme by measuring the unfolding length and key force of the molecule in real time, so as to evaluate the drugability of the ribozyme candidate drug at the single molecule level.

Description

Technical Field

[0001] This invention relates to a single-molecule mechanical method for detecting ribozyme isomers and evaluating the druggability of ribozyme candidate drugs. It uses single-molecule technology to perform mechanical detection and enzyme activity measurement on ribozymes. In particular, it uses single-molecule mechanical technology to control the folding state of ribozymes and measure the unfolding length and key forces of ribozymes in real time, thereby resolving the isomers of ribozymes. This invention belongs to the fields of medicinal chemistry and biology, as well as the field of nucleic acid drugs. Background Technology

[0002] Over the past three decades, experimental techniques for applying forces to biological samples have advanced to the point where individual biomolecules can be monitored and precisely controlled in real time. Currently, magnetic tweezers, optical tweezers, and atomic force microscopy are the mainstream single-molecule mechanics techniques. Single-molecule force spectroscopy, with its high spatiotemporal resolution, has been widely used to study the behavior of biomolecules at different force levels, including nucleic acid structures, the mechanics of polymers, the dynamics of motor proteins, protein folding and unfolding pathways, ligand-receptor binding, and the binding and dissociation of biomolecular complexes involved in mechanobiological processes.

[0003] Single-molecule magnetic tweezers, a representative technique in single-molecule force spectroscopy, apply mechanical loads at a level adapted to the structure and function regulation of biomolecules, enabling the detection of the random, fine dynamic behavior of individual molecules in solution. Their resolution can resolve the spatial changes of individual nucleotides on a biological reaction timescale, and the mechanical measurement range covers common intermolecular interactions such as hydrogen bonds, metal coordination bonds, and hydrophobic interactions. Chinese patent CN111254144A discloses a molecular ruler hairpin structure and a method for measuring the spatial scale accuracy of single-molecule magnetic tweezers. This method constructs a hairpin structure that generates pause signals under magnetic force as a molecular ruler. By calculating the difference between adjacent pause signals and performing a conversion from length to base pairs, the conversion coefficient is accurately calculated and compared with theoretical values, thus serving as a basis for evaluating the accuracy of single-molecule magnetic tweezers in spatial scale measurements. (Li Ning et al.) The dynamics of triple-stranded nucleic acid formation in artificial telomeres were revealed using single-molecule magnetic tweezers, providing a tool for the kinetic study of nucleic acid structures and the development of nucleic acid drug technology. Telomeres carrying repetitive sequences terminate with single-stranded protrusions. G-rich protrusions can fold and bind to the major groove of their upstream double strands to form antiparallel triple-stranded structures. Therefore, telomere triplets can protect open chromosome ends from damage repair mechanisms. A protective tether strategy was developed with the aid of click chemistry and branching polymerase chain reaction for single-molecule mechanical manipulation of nucleic acids with open ends. The probability of telomere triple-stranded structure formation is low (5%), which can pause the passive unwinding process of nucleic acids. The mechanically stable triple strands formed in telomere DNA can withstand a mechanical load of 20 piconels for several seconds in physiological buffer. Furthermore, the mechanical manipulation assisted by the protective tether strategy can directly disrupt the interaction between the third strand and the other two double strands in triple-stranded nucleic acids, allowing for the exploration of the pharmacological mechanisms and druggability of nucleic acid drugs based on triple-stranded structures. (Li Xu et al.) A method for directly measuring telomere nucleic acid length was developed, and the molecular dynamics of the telomere repeat sequence binding factor TRF1 protein at the telomere DNA replication fork were elucidated. This study directly obtained telomeres from human leukemia cells and then used single-molecule force spectroscopy to measure the absolute length of the telomeres, examining cellular senescence characteristics. TRF1 is the core subunit of the telomere structure protection complex Shelterin, playing a crucial role in structural maintenance and functional protection during telomere organization and replication. The study used single-molecule magnetic tweezers to investigate the compression and decompression effects of TRF1 dimers on telomeres. TRF1 was released from compressed telomere tissue through mechanical loading within ~20 seconds, and the release heterogeneity was measured, revealing a negative correlation between the number and size of heteroloops. Furthermore, measurements of the molecular dynamics of TRF1 at the telomere DNA replication fork showed a protein-nucleic acid binding energy of 11 kJ / L. B Furthermore, TRF1 can regulate the forward and backward pace of DNA replication forks at a critical force of ½ mechanical load and on a 10-second scale, maintaining the telomere replication fork in a temporarily open state and providing a time window for cellular regulation of replication fork homeostasis. The results also reveal the kinetics of TRF1 and the mechanism by which it promotes efficient telomere DNA replication, providing a technological platform for the diagnosis and treatment of telomere-related diseases and the discovery of nucleic acid-targeted drugs.

[0004] Single-molecule mechanics methods can dynamically analyze the interactions between biomolecules in real time at the single-molecule level, revealing the dynamic mechanisms of biomolecules, exploring precise quantification techniques for single-molecule conformational information, and directly measuring the temporal information of drug intervention interactions with biomolecules, providing novel indicators for drug efficacy evaluation. (Liang Lin et al.) The binding kinetics of the CXXC domain of the epigenetic enzyme MLL1 protein to CpG islands on long DNA was detected using single-molecule magnetic tweezers, solving the problem that current methods cannot detect the dynamic interactions between proteins and nucleic acids on dense CpG motifs using short DNA fragments. CpG islands recruit MLL1 through the CXXC domain to regulate chromatin structure and gene expression. The amino acid motif of CXXC also plays a crucial role in reading specific nucleic acid information in the structure and function of MLL1, serving as a target for drug design. Under the mechanical load provided by single-molecule magnetic tweezers, the nucleic acid-protein complex undergoes unwinding. By monitoring the unwinding time dynamics of the nucleic acid-protein complex in real time, the spatiotemporal information of the unwinding of individual CXXC-CpG complexes can be accurately detected. Furthermore, when specific small molecule chemical drugs, such as dimethyl fumarate, are added, their response curves can be obtained by monitoring the dosage added at the time of unwinding events in real time, thereby quantifying the inhibitory effect of the small molecule chemical drug on the CXXC-DNA complex. This further demonstrates the capability and feasibility of single-molecule mechanical methods in drug discovery and screening, as well as in chemical biology research. Meanwhile, high efficiency is an indispensable characteristic of biocoupling, a crucial method for studying the interactions of macromolecules such as proteins and nucleic acids. US Patent US11505800 B2 and Chinese Patent CN109750055B disclose a method for improving protein-nucleic acid biocoupling efficiency using specific alpha helical cables based on genetic codon expansion technology. This method involves constructing a fusion recombinant expression plasmid carrying a cable of non-natural amino acids and a protein of interest, purifying and expressing it, and then linking it to a coupling group on a nucleic acid substrate. This provides a controllable and reaction-friendly protein surface nanoenvironment for the non-natural amino acids, avoiding the harsh nanoreaction environment shaped by the complex structure, charge, and polarity of the protein of interest surface, thus improving the biocoupling efficiency between proteins and nucleic acids. Improving the protein-nucleic acid biocoupling efficiency is particularly important for measuring the role of drugs in regulating the activity and function of biomolecules, and has significant application value and engineering significance in the interdisciplinary fields of medicinal chemistry and biology. Meanwhile, Liang Lin et al. A single-molecule method has been developed to enable multiplex analysis of nucleic acid-protein complexes using single-molecule magnetic tweezers. When a double-stranded nucleic acid with multiple protein-binding sites unwinds, strand dissociation pauses at the protein-binding site. Single-molecule mechanics can be used to directly measure the binding probability and dissociation time of the protein at specific nucleic acid sites. Studies have shown that, taking the CXXC domain of the epigenetic enzyme TET1 protein as an example, in A- or T-rich DNA, TET1 CXXC can bind multiple CpG motifs. At nanometer resolution, it can be determined that TET1 CXXC prefers CpG motifs flanked by G or C. Simultaneously, TET1 CXXC can distinguish five CpG clusters within the binding motifs on CpG islands. Therefore, single-molecule techniques can probe site-specific nucleic acid-protein interactions and reveal their molecular dynamics. The feasibility of single-molecule multiplex mapping analysis will contribute to the precise quantification and understanding of nucleic acid-protein interactions. Furthermore, epigenetics, such as the dynamic interactions between DNA methylation and demethylation, plays a crucial role in key cellular events. The enzyme activity of CpG sites with cytosine methylation or demethylation is influenced by CpG density, methylation state, and flanking sequences. Multiplex analysis using single-molecule methods is significant for studying the mechanisms by which related enzymes recognize target DNA and for the development of epigenetic enzyme targets. (Ma Xiaofeng et al.) The interaction between the PHD3-Bromo domain of the epigenetic enzyme MLL1 protein and the methylated histone H3K4me3 was revealed using single-molecule magnetic tweezers within a parallel force path of nucleic acids, achieving single-molecule force spectroscopy measurements of protein-protein interactions. The interaction between PHD3-Bromo and K4-methylated histone H3 is a key epigenetic event in leukemia development. However, protein aggregation caused by specific or non-specific factors often occurs in the detection of protein-protein interactions. Using single-molecule magnetic tweezers and a parallel force path strategy with nucleic acids allows negatively charged DNA to act as a linker, thus preventing protein aggregation in the interaction construct. The results show that the key force disrupting protein-protein interactions is 12 piconewtons, significantly higher than the 3 piconewtons key force for DNA extraction from nucleosomes. Therefore, single-molecule mechanics methods can accurately measure protein-protein interactions, and their high precision and specificity make it possible to elucidate the mechanisms of intermolecular interactions, further promoting the discovery of protein-targeted drugs.

[0005] Unfolding experiments in single-molecule mechanics mode enable the study of the physicochemical properties of nucleic acids, from the thermodynamics of double-strand formation to the folding of secondary and tertiary structures. RNA, in particular, exhibits highly complex dynamics due to the multiple long-lasting intermediate states that occur during molecular folding. Single-molecule mechanics techniques allow us to derive key thermodynamic and kinetic parameters of the RNA folding process, thereby characterizing intermediate products and further understanding the formation pathways of native structures. In a single unfolding experiment, nucleic acid molecules are mechanically stretched apart under an applied force. This allows for the measurement of the force-distance curve, which displays a sequence-related zigzag pattern.

[0006] Nucleic acids, as important components of biological macromolecules, are also well-defined drug targets. Nucleic acid drugs are currently a hot topic and crucial link in pharmaceutical research and development, possessing clear and broad application prospects and extremely high clinical value. Taking ribozymes as an example, ribozymes are catalytically active RNA molecules, a class of naturally occurring RNA motifs that play a central role in the replication of RNA viruses or viroids, the regulation of messenger RNA stability, and protein synthesis. Among them, hammerhead ribozymes are the most widely reported and studied members of this family. Studies have discovered many new variants of this ribozyme in the genomes of organisms from all living things, performing numerous previously unknown functions. Therefore, the study of ribozyme structure and function, especially isoform conformations, has significant and far-reaching implications in biology. However, to date, no methods for detecting ribozyme isoforms at the single-molecule level have been reported.

[0007] [1]Li N, Wang J, Ma K, et al. The dynamics of forming a triplex in anartificial telomere inferred by DNA mechanics [J]. Nucleic acids research, 2019, 47(15): e86. [2]Li X, Wang M, Zheng W, et al. Dynamics of TRF1 organizing a singlehuman telomere [J]. Nucleic acids research, 2021, 49(2): 760-775. [3]Liang L, Ma K, Wang Z, et al. Dynamics and inhibition of MLL1 CXXCdomain on DNA revealed by single-molecule quantification [J]. Biophysicaljournal, 2021, 120(16): 3283-3291. [4]Liang L, Wang Z, Qu L, et al. Single-molecule multiplexed profiling of protein-DNA complexes using magnetic tweezers [J]. The Journal of biological chemistry, 2021, 296: 100327. [5]Ma Summary of the Invention

[0008] This invention provides a single-molecule method for detecting ribozyme isomers, characterized by high throughput, high efficiency, and high resolution. Specifically, this invention utilizes a constant magnetic field provided by single-molecule magnetic tweezers to detect the folding length and key forces of ribozymes by controlling the distance between a magnet in the magnetic field and a mechanically manipulated structure. This method is particularly suitable for druggability assessment of ribozyme drug candidates.

[0009] The technical solution of this invention is:

[0010] This invention first designs two 668-base-pair double-stranded DNA strands modified with biotin-16-dUTP and digoxigenin-11-dUTP, namely a 5' end modified strand and a 3' end modified strand. Next, two isolation strands with overhanging ends are formed by annealing four oligonucleotides, namely a 5' end isolation strand and a 3' end isolation strand. A DNA flanking sequence is synthesized at the end of the ribozyme for recognition and ligation by the ligase. Finally, the 5' end modified strand, the 3' end modified strand, the 5' end isolation strand, the 3' end isolation strand, and the ribozyme are efficiently ligated under the action of DNA ligase.

[0011] This invention provides a method for detecting ribozyme isomers based on single-molecule mechanical technology, characterized by comprising the following steps:

[0012] 1) Based on the highly conserved active center and specific target molecule sequence of the tertiary structure of the classic hammerhead ribozyme, a ribozyme structure that can be correctly folded and has enzymatic activity was designed and synthesized.

[0013] 2) A single-stranded RNA substrate was synthesized based on the sequence of the ribozyme substrate target molecule;

[0014] 3) Based on the experimental architecture of the single-molecule mechanical platform, we designed and synthesized 5'-end modified cables and 3'-end modified cables doped with biotin and digoxigenin, and annealed them to form 5'-end isolated cables and 3'-end isolated cables with overhanging ends;

[0015] 4) Based on nucleic acid ligation reactions, ribozymes, modified 5' end modified ligands and 3' end modified ligands, and 5' end isolated ligands and 3' end isolated ligands with overhanging ends are efficiently ligated in steps to form DNA-RNA-DNA structures for single-molecule mechanical manipulation.

[0016] 5) Using single-molecule magnetic tweezers, the mechanical properties of the designed and constructed DNA-RNA-DNA molecules are detected, the unfolding length and key forces are measured in real time, and the isomers of ribozymes are identified;

[0017] As mentioned above, we have achieved the detection of ribozyme isomers using single-molecule mechanical techniques.

[0018] The ribozyme described in step 1) is 54 nucleotides in length, obtained by chemical synthesis, and is divided into five parts: Helix I, Helix II, Helix III, and core domains Domain I and Domain II, forming an overall "Y" shape.

[0019] The RNA substrate described in step 2) is complementary to the ribozyme bases, has a length of 15 nucleotides, and is obtained by chemical synthesis.

[0020] Step 3) The biotin-modified and digoxigenin-modified 5'-end modified ligands and 3'-end modified ligands are synthesized by using specific primers on the pBluescript II SK(+) plasmid, using dATP, dCTP, dGTP, dTTP, digoxigenin-11-dUTP, and biotin-16-dUTP as raw materials, and under the action of Taq DNA polymerase through polymerase chain reaction. The four oligonucleotides are annealed to form 5'-end isolated ligands and 3'-end isolated ligands with overhanging ends, respectively.

[0021] The ribozyme, modified 5' end modified ligand and 3' end modified ligand, and 5' end isolation ligand and 3' end isolation ligand with overhanging ends described in step 4) are ligated by T4 DNA ligase.

[0022] Step 5) describes the single-molecule magnetic tweezers as an important tool in the single-molecule mechanics method. A pair of permanent magnets provides a uniform and constant magnetic field strength. When the target molecule is stretched by an external force in the magnetic field, its spatial structure changes from a folded state to a linear state in sequence. When the force gradually decreases, it returns to the folded state from the linear state. The measured unfolding length and key force correspond to the mechanical properties of different structural domains of the ribozyme, thereby analyzing the isomerism phenomenon of the ribozyme.

[0023] This invention provides a single-molecule method for detecting ribozyme isomers. By measuring the force-stretch curve, the unfolding length and key forces of the ribozyme are obtained, enabling real-time analysis of the conformational dynamics of the ribozyme at both the millisecond and nanometer scales, thus resolving ribozyme isomers. The application of single-molecule technology overcomes the shortcomings of traditional methods in structural analysis, making isomer detection possible and allowing for the evaluation of the druggability of nucleic acid drugs at the single-molecule level, thus better supporting their use in biological research and clinical applications. Attached Figure Description

[0024] Figure 1 Schematic diagram of mechanical manipulation of a structure.

[0025] Figure 2 Experimental diagram of force slope under the mechanical manipulation mode of magnetic tweezers.

[0026] Figure 3 Typical molecular mechanical curves.

[0027] Figure 4 Statistical distribution and Gaussian fitting curve of fold length and key forces in nuclear enzyme digestion.

[0028] Figure 5 Statistical distribution and Gaussian fitting curve of unfolding length and key force when ribozymes bind to substrates. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. Experimental procedures not specifically described in the embodiments follow conventional procedures or reaction conditions recommended in product instructions. Unless otherwise specified, general equipment, materials, and reagents used in the embodiments can be purchased commercially.

[0030] The present application will be further illustrated below with specific embodiments.

[0031] Example 1: Design of mechanically controlled structures

[0032] 1. Preparation of 5' end modified cables and 3' end modified cables

[0033] 1.1 Polymerase chain reaction of 5'-terminal modified Lasso and 3'-terminal modified Lasso

[0034] The pBluescript II SK(+) plasmid was subjected to a polymerase chain reaction involving biotin / digoxigenin modification. The reaction system is as follows:

[0035] 1 μl of 10 μM biotin / digoxin laccoside upstream primers, as shown in SEQ ID NO.1 and SEQ ID NO.3, respectively (primers purchased from Genewiz).

[0036] 1 μl of 10 μM biotin / digoxinraxol downstream primers, as shown in SEQ ID NO.2 or SEQ ID NO.4, respectively (primers purchased from Genewiz).

[0037] 1 μl 10 mM dATP (Catalog No.: 4026Q, Baori Biotechnology Co., Ltd.);

[0038] 1 μl 10 mM dGTP (Catalog No.: 4027Q, Baori Biotechnology Co., Ltd.);

[0039] 1 μl 10 mM dCTP (Catalog No.: 4028Q, Baori Biotechnology Co., Ltd.);

[0040] 0.9 μl 10 mM dTTP (Catalog No.: 4029Q, Baori Biotechnology Co., Ltd.);

[0041] 1 μl 1 mM Bio-16-dUTP (Catalog No.: 11093070910, Roche) / 1 μl 1 mM Dig-11-dUTP (Catalog No.: 11093088910, Roche).

[0042] 1 μl 5 ng / ml pBluescript II SK(+) plasmid (Sangon Biotech Co., Ltd.);

[0043] 0.25 μl Taq DNA polymerase (Catalog No.: EP0402, Thermo Fisher Scientific);

[0044] 3 μl of 25 mM MgCl2 solution;

[0045] 5 μl 10×Taq KCl buffer;

[0046] 33.85 μl of pure water.

[0047] The temperature-controlled cycle for the polymerase chain reaction is 95°C. o C remained unchanged for 3 minutes, then decreased by -0.1. o The speed of C / 8 seconds drops to 72. o C, in 72 o C holds for 3 minutes, finally ending at 4. o C.

[0048] The product was purified using the Cycle Pure kit (catalog number: D6492-02, Omega). The lengths of the 5' and 3' modified filaments were 668 bp.

[0049] 1.2 Enzyme digestion of 5'-terminal modified Lasso and 3'-terminal modified Lasso

[0050] The 5' end modified Lasso enzyme digestion reaction system is as follows:

[0051] 56 μl of the 5' end modified laccase obtained in the first step (125.0 ng / μl);

[0052] 18 μl 10×CutSmart buffer;

[0053] 17.5 μl BbvCI restriction enzyme (Catalog No.: R0601S, Neb Biosciences);

[0054] 88.5 μl of pure water.

[0055] After mixing, divide the mixture evenly into four reaction tubes.

[0056] The 3' end modified Lasso enzyme digestion reaction system is as follows:

[0057] 44 μl of the 3' end modified lassyl obtained in the first step (134.2 ng / μl);

[0058] 13.5 μl 10×CutSmart buffer;

[0059] 3.5 μl PpuMI restriction enzyme (Catalog No.: R0506L, Neb Biosciences);

[0060] 74 μl of pure water.

[0061] After mixing, divide the mixture evenly into three reaction tubes.

[0062] The temperature control program for polymerase chain reaction is: 37°C. o Keep C for 12 hours.

[0063] The product was purified using the cycle-pure kit to obtain 5'-terminal modified LASIK and 3'-terminal modified LASIK.

[0064] 2. Preparation of 5' end isolation cables and 3' end isolation cables

[0065] 2.1 Annealing reaction

[0066] The annealing reaction system for the 5' end isolation cable is as follows:

[0067] 8 μl 50 mM nucleic acid strand as shown in SEQ ID NO.5;

[0068] 8 μl 50 mM nucleic acid strand as shown in SEQ ID NO.6;

[0069] 4 μl 5×DNA annealing buffer.

[0070] The annealing reaction temperature control program is: 98 o C remains constant for 3 minutes, at -0.1 o Cool to 25°C at a rate gradient of 8 seconds. o C, 25 o Maintain C for 3 minutes, and finally stop at 12 ºC.

[0071] The annealing reaction system for the 3' end isolation cable is as follows:

[0072] 8 μl 50 mM nucleic acid strand as shown in SEQ ID NO.7;

[0073] 8 μl 50 mM nucleic acid strand as shown in SEQ ID NO.8;

[0074] 4 μl 5×DNA annealing buffer.

[0075] The annealing reaction temperature control program is: 98 o C remains constant for 3 minutes, at -0.1 o Cool to 25°C at a rate gradient of 8 seconds. o C and hold for 3 minutes, finally stopping at 12. o C.

[0076] 2.2 Phosphorylation reaction

[0077] The phosphorylation reaction system for the 5' end isolation cable and the 3' end isolation cable is as follows:

[0078] 8.5 μl of annealed product;

[0079] 1 μl 10× T4 PNK buffer;

[0080] 0.5 μl T4 phosphokinase.

[0081] The phosphorylation temperature control program is: 37°C o C remains unchanged for 90 minutes, 70 o C remains constant for 5 minutes, at -0.1 o Cool to 25°C at a rate gradient of 8 seconds. o C and hold for 3 minutes, finally stopping at 12. o C.

[0082] 3. Phosphorylation of ribozymes

[0083] The reaction system is as follows:

[0084] 8.5 μl of RNA ribozyme with the sequence shown in SEQ ID NO.9;

[0085] 1 μl 10×T4 PNK buffer;

[0086] 0.5 μl T4 phosphokinase.

[0087] The phosphorylation temperature control program is: 37°C o Keep C for 30 minutes.

[0088] 4. Ligation of 5' end modified ligand, 3' end modified ligand, 5' end isolation ligand, 3' end isolation ligand and ribozyme molecule.

[0089] The connection reaction system is as follows:

[0090] 2.8 μl 5´ end modified cable;

[0091] 2.2 μl 3´ end modified ligand;

[0092] 1 μl 5´ end isolation cable;

[0093] 1 μl 3´ end isolation cable;

[0094] 10 μl RNA ribozyme;

[0095] 2 μl 10×T4 DNA ligation reaction buffer;

[0096] 1 μl T4 DNA ligase (Catalog No.: M0202S, NEB).

[0097] The connection to the reaction temperature control program is: 16o Keep C for 12 hours.

[0098] Thus, the structures used for single-molecule mechanical manipulation were obtained, such as... Figure 1 As shown.

[0099] Example 2: Application of single-molecule magnetic tweezers in the detection of DNA-RNA-DNA molecules

[0100] The sample cell preparation process is as follows: Take two coverslips measuring 60 mm × 24 mm × 0.13 mm. Punch holes at both ends of one of the coverslips using a TC-169 multi-functional grinding and cutting machine. Place both coverslips on a slide holder and clean them sequentially with deionized water, detergent, isopropanol solution, and 75% ethanol solution. Finally, dry the surface with nitrogen gas. Evenly coat the surface of the un-punched coverslip with 3 μm monodisperse polystyrene microspheres stored in a 0.1% nitrocellulose membrane solution for use as reference spheres in subsequent experiments. Stack the two coverslips and a double-layer sealing film prepared using a metal mold, with the un-punched coverslip at the bottom, the two sealing films in the middle, and the perforated coverslip at the top to form the sample cell. Place the cell in a metal bath and heat to solidify. First, add 70 μl of 0.1 mg / ml anti-digoxin antibody (catalog number: 11214667001, Sigma-Aldrich China) in PBS solution to the sample cell and incubate at room temperature for 1 hour. Then, wash the sample cell three times with PBS buffer, add 70 μl of 5 mg / ml bovine serum albumin (BSA) solution and block at room temperature for 12 hours. Finally, wash with PBS buffer and fill the sample cell, and store at 4 °C for later use.

[0101] The mechanical control mode of the magnetic tweezers device is: force slope experiment.

[0102] The sample system is as follows: 3 μl DNA-RNA-DNA molecules and 30 μl M270 magnetic beads (catalog number: 65305, Invitrogen) are incubated in an ice bath for 10 minutes to allow the biotin-modified ends to bind to the magnetic beads and form ribozyme beads. Then, the ribozyme beads are added to the sample cell and incubated statically to allow the sample to bind to the anti-digoxigenin antibody on the coverslip below and be fixed on the surface of the coverslip. Finally, the unbound or floating magnetic beads on the top of the coverslip are rinsed with working buffer at an appropriate flow rate.

[0103] In this embodiment, molecular dynamics are observed in real time using single-molecule magnetic tweezers, and the data generated by the magnetic tweezers is analyzed and processed to obtain the relationship between the force, time, and stretching length of the DNA-RNA-DNA molecule, such as... Figure 2-4 As shown. Figure 2This is a schematic diagram of the mechanical manipulation mode of magnetic tweezers on ribozyme spheres. When no force is applied, the ribozyme molecules on the ribozyme spheres are in a folded state. As the force is applied and increased, the molecules gradually unfold under the action of external force and become a linear structure. Figure 3 It is a typical line of molecular signal; during mechanical manipulation, the target molecule generates an unfolding signal. Figure 4 The graphs show the unfolding length and key unfolding forces of the molecule, along with their Gaussian distribution fitting curves. The left graph shows the working buffer containing magnesium ions under experimental conditions, while the right graph shows the working buffer without magnesium ions under experimental conditions. The top graph shows the unfolding length, and the bottom graph shows the unfolding force.

[0104] Example 3: Application of single-molecule magnetic tweezers in the detection of ribozyme isomers

[0105] The sample cell preparation process in this embodiment is the same as in Embodiment 2.

[0106] The mechanical control mode of the magnetic tweezers device is the same as in Example 2.

[0107] The sample system is as follows: 3 μl DNA-RNA-DNA molecules are bound to 3 μl ribozyme substrate RNA (sequence SEQ ID NO.10) and incubated with 30 μl M270 magnetic beads in an ice bath for 10 minutes to allow the biotin-modified ends to bind to the magnetic beads and form ribozyme beads. Then, the ribozyme beads are added to the sample cell and incubated statically to allow the sample to bind to the anti-digoxigenin antibody on the coverslip below and be fixed on the surface of the coverslip. Finally, the unbound or floating magnetic beads on the top of the coverslip are rinsed with working buffer at an appropriate flow rate.

[0108] In this embodiment, the dynamics of molecules bound to the substrate are observed in real time using single-molecule magnetic tweezers, and the data generated by the magnetic tweezers is analyzed and processed to obtain the relationship between the force and the change in unfolding length of DNA-RNA-DNA molecules after binding to the substrate, thereby resolving the isomers of ribozymes. Figure 5 The figures show the histogram and Gaussian distribution fitting curve of the unfolding length and key unfolding force of the molecule after binding to the substrate. The left figure shows the working buffer containing magnesium ions under experimental conditions, while the right figure shows the working buffer without magnesium ions under experimental conditions. The top figure shows the unfolding length, and the bottom figure shows the unfolding force.

[0109] The foregoing has described some embodiments of the present invention in detail, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should fall within the patent coverage of the present invention.

[0110] SEQUENCE LISTING

[0111] <110> Nankai University

[0112] <120> A single-molecule method for detecting ribozyme isomers

[0113] <130> 202301

[0114] <160> 1

[0115] <170> PatentIn version 3.5

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[0175] aagggucucu guaca 15

Claims

1. A method for detecting ribozyme isomers based on single-molecule technology, characterized in that: The unfolding state of ribozymes was controlled using single-molecule mechanics methods, and the conformation of ribozyme isomers was detected by measuring the unfolding length and key forces. The ribozyme is a chemically synthesized RNA with a full length of 54 nucleotides. The 5' end of the ribozyme is connected to a 5' end modification cable via a 5' end isolation cable, and the 3' end of the ribozyme is connected to a 3' end modification cable via a 3' end isolation cable. Both the isolation cable and the modification cable are DNA, forming a DNA-RNA-DNA mechanical control structure. The mechanically manipulated structure is formed by connecting a 5' end modified cable, a 5' end isolation cable, a ribozyme, a 3' end isolation cable, and a 3' end modified cable via a DNA ligase. The 5' end modified Lasso and the 3' end modified Lasso are derived from plasmid DNA. The 5' end modified Lasso is modified with biotin, and the 3' end modified Lasso is modified with digoxigenin. Both are 668 base pairs in length. The 5' end isolation cable and the 3' end isolation cable are formed by annealing two chemically synthesized oligonucleotide chains, respectively. The lengths of the oligonucleotide chains forming the 5' end isolation cable are 35 nucleotides and 39 nucleotides, respectively, and the lengths of the oligonucleotide chains forming the 3' end isolation cable are 43 nucleotides and 53 nucleotides, respectively. The detection was performed in a liquid environment. The ribozyme unfolded under the stretching of single-molecule magnetic tweezers. The isomers of the ribozyme were analyzed based on the statistical distribution of unfolding length and key forces measured in all unfolding single-molecule events. Gaussian fitting was used to characterize the characteristic length and key forces of the unfolding of the ribozyme isomers. The activity of the ribozyme was measured by adding a single-stranded RNA substrate to the buffer solution. The ribozyme bound to the substrate, and the activity of the ribozyme was determined by the change in the statistical distribution of unfolding length and key forces compared to when no substrate was added. If the expected Gaussian peak disappeared in the statistical distribution, it proved that the ribozyme was active.

2. The method according to claim 1, characterized in that: The mechanically manipulated structure is formed by linking T4 DNA ligase in a predetermined direction from the 5' end to the 3' end, consisting of a 5' end modified ligand, a 5' end isolation ligand, a ribozyme, a 3' end isolation ligand, and a 3' end modified ligand.

3. The method according to claim 1, characterized in that: The 5' end modified cable is modified with biotin-16-dUTP, and the 3' end modified cable is modified with digoxin-11-dUTP.

4. The method according to claim 1, characterized in that: Includes the following steps: The design and amplification of 5' and 3' modified Lasso from pBluescript II SK(+) plasmid using polymerase chain reaction were performed. The reaction program was as follows: 95℃, 3 min; 95-72℃, decreasing the temperature by 0.1℃ every 8 seconds to 72℃; 72℃, 3 min; stored at 4℃. The 5'-terminal modified Lasso and the 3'-terminal modified Lasso were digested with restriction endonucleases BbvCI and PpuMI. The reaction program was 37°C for 12 hours. Annealing was used to prepare 5'-end and 3'-end isolation cables, followed by phosphorylation. The annealing program was as follows: 98°C for 3 minutes, then 98°C to 25°C, decreasing by 0.1°C every 8 seconds to 25°C; 25°C for 3 minutes; and stored at 12°C. The phosphorylation program was as follows: 37°C for 90 minutes, 70°C for 5 minutes, then 70°C to 25°C, decreasing by 0.1°C every 8 seconds to 25°C; 25°C for 3 minutes; and stored at 12°C. The prepared 5'-terminal modified ligand, 5'-terminal isolated ligand, 3'-terminal modified ligand, 3'-terminal isolated ligand and ribozyme were ligated with T4 DNA ligase to obtain the mechanically manipulated structure. The reaction program was: 16℃, 12 hours.

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