Engineering C-Dz and construction method and application thereof

Abstract

The invention relates to an engineered C-Dz and a construction method thereof, the engineered L-Dz is obtained by inserting a fluorescence coding region and a time coding region into L-Dz, the length of a detection single arm of the engineered L-Dz is set to be 5-12nt, and the engineered C-Dz is obtained after cyclization; when the length of the engineered C-Dz detection single arm constructed by the construction method is 5-12nt, the catalytic efficiency is relatively high, and particularly, the catalytic efficiency is the highest at 8nt; the invention relates to a construction method of a TIME-FLUO bar code. The construction method comprises the following steps: carrying out rolling circle amplification on engineered C-Dz to obtain a two-dimensional TIME-FLUO bar code containing time and fluorescence signals; when the engineered C-Dz comprises at least two engineered C-Dz with different time coding region lengths, the spectrums of the hairpin probe fluorophores matched with the fluorescence coding regions corresponding to the engineered C-Dz are not overlapped; according to the application of the TIME-FLUO bar code in preparation of the polyvirus detection kit, an extensible framework is established for high-dimensional molecular diagnosis, and the application prospect is good.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an engineered C-Dz and its construction method and application. Background Art

[0002] DNA barcoding technology is a biotechnology that identifies and encodes targets based on the characteristics of DNA molecules. Its core principle is to use the programmability of DNA sequences and the high specificity of the base sequence of DNA chains to design short-chain DNA sequences. Different sequences can also be combined with fluorescent markers (such as fluorescent dyes and quantum dots) to generate a large number of coding combinations, giving different target molecules (such as proteins, nucleic acids, pathogens, etc.) unique "molecular labels", thereby realizing high-throughput, multi-target parallel detection.

[0003] DNAzyme is a type of single-stranded DNA molecule with catalytic function, which can recognize and bind substrates through a specific spatial conformation and bind to substrates in the presence of metal ion cofactors (such as Mg 2+ 、Zn 2+ In the presence of phosphodiesterases (e.g., phosphodiesterases), DNAzymes efficiently catalyze reactions such as RNA cleavage, ligation, or chemical modification. Their core function relies on a catalytically active region and substrate recognition region composed of deoxyribonucleotides, achieving specific substrate recognition through complementary base pairing. Compared to traditional protein enzymes, DNAzymes offer advantages such as flexible design, high thermal stability, low synthesis costs, and strong programmability. They are widely used in biosensing, gene regulation, disease diagnosis, and treatment. By rationally designing their sequence and structure, their catalytic activity, selectivity, and environmental responsiveness can be manipulated, making them important tool molecules in synthetic biology and nanotechnology.

[0004] Circular DNAzymes (C-Dz) are typically constructed through the precise circularization of linear DNAzymes (L-Dz). Specifically, complementary short nucleotide sequences (such as sticky ends or hairpin structures) are introduced at both ends of the linear DNAzyme. Enzymatic ligation (e.g., with T4 DNA ligase) or self-assembly promotes pairing and covalently closed circular structures. During the cyclization process, reaction conditions (such as temperature, ion concentration, and ligase dosage) must be optimized to ensure efficient ring closure while avoiding residual linear intermediates. The formation of the circular structure not only eliminates the free energy difference at the linear DNAzyme termini but also enhances structural stability through topological constraints, making it more resistant to exonuclease degradation. Furthermore, cyclization may optimize the local conformation of the functional domain, promoting specific binding of the catalytic core to the substrate, thereby enhancing cleavage activity. The successful preparation of cyclic C-Dz is typically verified by gel electrophoretic mobility differences or sequencing, ultimately yielding a circular DNAzyme that combines efficient catalytic performance with structural stability.

[0005] If the cleavage rate of cyclic C-Dz is significantly reduced, it may lead to multiple negative effects. First, in catalytic applications (such as RNA cleavage or biosensing), the reaction efficiency will decrease, extending the time required to reach the expected substrate conversion rate, which may affect the sensitivity of real-time detection or the timeliness in treatment scenarios. Second, in dynamic biological systems (such as the intracellular environment), the slow catalytic rate may not be able to effectively compete for the degradation of the substrate or interact with other biological molecules, resulting in functional failure. Therefore, optimizing the conformational design of the ring structure or the length of the functional region and balancing stability and catalytic activity are the keys to improving its effectiveness. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention proposes an engineered C-Dz and its construction method and application.

[0007] The technical solution of the present invention is:

[0008] A method for constructing an engineered C-Dz comprises inserting a fluorescence coding region and a time coding region into L-Dz to obtain the engineered L-Dz, setting the length of the L-Dz detection single arm to 5-12 nt, and cyclizing the engineered L-Dz to obtain the engineered C-Dz.

[0009] Furthermore, the length of the L-Dz detection single arm is 8 nt.

[0010] Furthermore, the time coding region is arranged downstream of the engineered C-Dz recognition region, and the fluorescence coding region is arranged downstream of the time coding region.

[0011] Furthermore, the time coding region is a repeated thymine sequence.

[0012] Furthermore, the construction method of the engineered C-Dz is specifically as follows:

[0013] S1. Preparation of L-Dz: Connect the catalytic core region, detection arms, fluorescence coding region, and time coding region of L-Dz to form an engineered L-Dz;

[0014] S2. Circularization to form engineered C-Dz: First, phosphorylate the engineered L-Dz. Mix the phosphorylated L-Dz, primers, and DEPC water, then heat at 95°C for 5 minutes. Then, anneal to room temperature. Then, add T4 DNA ligase and T4 DNA ligase buffer to the system, and incubate overnight at 16°C to obtain a mixture solution containing the engineered C-Dz.

[0015] S3. Digestion of uncyclized engineered L-Dz: Heat and denature the engineered C-Dz mixture solution prepared in S2 to inactivate T4 DNA ligase. Then add Exonuclease I, Exonuclease I buffer, and Exonuclease III to digest the uncyclized engineered L-Dz overnight. Heat and incubate to inactivate enzyme activity to obtain engineered C-Dz, which is then stored at -20°C.

[0016] The engineered C-Dz was constructed according to the construction method.

[0017] A method for constructing a TIME-FLUO barcode, wherein the engineered C-Dz is subjected to rolling circle amplification to generate a two-dimensional TIME-FLUO barcode containing time and fluorescence signals.

[0018] Furthermore, the engineered C-Dz comprises at least two engineered C-Dz with different time coding region lengths.

[0019] Furthermore, the spectra of the fluorescent groups of the hairpin probes matched with the fluorescent coding regions corresponding to the engineered C-Dz with different time coding region lengths do not overlap, and the time coding regions with different lengths differ by 10-20 nt.

[0020] Application of the TIME-FLUO barcode constructed according to the construction method in the preparation of a multiple virus detection kit.

[0021] Furthermore, the multiple virus detection kit is used to detect respiratory viruses; the respiratory viruses are a combination of one or more of H1N1, FLUB, RSV and HRV.

[0022] Furthermore, the RNA sequence of H1N1 is shown as SEQ ID NO.5, the RNA sequence of FLUB is shown as SEQ ID NO.6, the RNA sequence of RSV is shown as SEQ ID NO.7, and the RNA sequence of HRV is shown as SEQ ID NO.8.

[0023] Compared with the prior art, the present invention has at least the following advantages:

[0024] 1. The present invention relates to an engineered C-Dz and a method for constructing the same. The engineered L-Dz is first obtained by inserting a fluorescence coding region and a time coding region into L-Dz, wherein the fluorescence coding region is complementary to a hairpin probe in a substrate, and the time coding region is a repeated thymine sequence. The length of the engineered L-Dz detection arm is then set to 5-12 nt, and the engineered L-Dz is cyclized to obtain the engineered C-Dz. The engineered C-Dz constructed by the construction method has a high catalytic efficiency when the detection arm length is 5-12 nt, and particularly has the highest catalytic efficiency when the length is 8 nt, which is nearly 7 times that of L-Dz.

[0025] 2. The present invention relates to a method for constructing a TIME-FLUO barcode. The engineered C-Dz can obtain a two-dimensional TIME-FLUO barcode containing time and fluorescence signals after rolling circle amplification. When the engineered C-Dz includes at least two engineered C-Dz with different time coding region lengths, the spectra of the hairpin probe fluorescent groups matched to the fluorescence coding regions corresponding to the engineered C-Dz do not overlap with each other, and the peak time of the fluorescence intensity of each engineered C-Dz during rolling circle amplification is different, thereby generating a dynamic TIME-FLUO barcode.

[0026] 3. The present invention relates to the use of TIME-FLUO barcodes in the preparation of multi-virus detection kits, which can be used for multi-detection of respiratory viruses. Specific TIME-FLUO two-dimensional barcodes are generated for one or more viruses, thereby identifying multiple targets that can be decoded from the signal. This establishes an expandable framework for high-dimensional molecular diagnosis and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0028] Figure 1 This is a schematic diagram of the preparation principle of CLOCK in Example 1 of the present invention;

[0029] Figure 2 This is a diagram showing the difference in cleavage between linear DNAzyme and circular DNAzyme under different exonuclease concentrations in Example 2 of the present invention; a schematic diagram showing the catalytic efficiency of circular DNAzyme and linear DNAzyme compared through model simulation;

[0030] Figure 3 The following describes the construction process of four engineered C-Dz molecules with different time coding region lengths in Example 3 of the present invention; the method for determining the peak time of the fluorescence curves of the four engineered C-Dz molecules; and the construction of a two-dimensional TIME-FLUO barcode by introducing a different fluorescent group into each engineered C-Dz molecule.

[0031] Figure 4 This is a schematic diagram of the amplification principle of the four-ring DNAzyme according to the embodiment of the present invention; six C-Dz with different lengths of single-arm detection regions were constructed and verified by gel electrophoresis; substrate cleavage verified the linear relationship between substrate concentration and fluorescence intensity;

[0032] Figure 5 This is the kinetic experiment of the six engineered C-Dz cleavages of Example 4 of the present invention; among them, the catalytic efficiency of the 8nt single-arm detection region CDz is the highest, which is nearly 7 times that of the 8nt L-Dz;

[0033] Figure 6 The engineered C-Dz of four functional regions of different lengths according to Example 5 of the present invention were constructed and the cleavage kinetics experiments were performed;

[0034] Figure 7 This is a schematic diagram of the multi-virus detection kit constructed in Example 6 of the present invention, and a diagram showing the specificity and sensitivity test results of the kit using four pseudoviruses and clinical samples. DETAILED DESCRIPTION

[0035] The present invention is described in further detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0036] This document provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods. All reagents and instruments used, unless the manufacturer is specified, are commercially available, conventional products and were prepared or used using conventional methods.

[0037] Sources

[0038] All oligonucleotides (Table 1) were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and purified by high-performance liquid chromatography (HPLC).

[0039] T4 DNA ligase, T4 PNK (T4 polynucleotide kinase), exonuclease I (Escherichia coli), exonuclease III (Escherichia coli), recombinant RNase inhibitor (ribonuclease inhibitor), dNTPs, phi29 DNA polymerase, phi29 DNA polymerase reaction buffer, magnesium sulfate (MgSO4) and recombinant albumin were purchased from New England Biolabs, USA.

[0040] 10× Tris-HCl (pH 7.4) was purchased from Sangon Biotech. Co., Ltd. (Shanghai, China).

[0041] QIAamp viral RNA mini kit (52904 / 52906) was purchased from QIAGEN, USA. All solutions were treated with DNase- and RNase-free water.

[0042] Experimental methods

[0043] Preparation of L-Dz and cyclization to obtain C-Dz: The total reaction system (20 μL) contained 1 μL of phosphorylated linear DNAzyme template (20 μM) and 1 μL of primer (20 M), mixed with 10 μL of DEPC water; the mixture was heated at 95°C for 5 minutes and then cooled by 1°C per minute to anneal to 25°C. Then, 1 μL of T4 DNA ligase and 1× T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT) were added to the system and incubated at 16°C overnight to form a circular DNAzyme. The mixture was denatured at 65°C for 10 minutes to inactivate T4 DNA ligase was added, followed by the addition of 2 μL of exonuclease I, 2 μL of exonuclease I reaction buffer, and 1 μL of exonuclease III to digest the linear DNA at 37°C overnight, incubated at 80°C for 20 minutes to inactivate the exonuclease I activity, and the obtained C-Dz was stored at -20°C.

[0044] DNAzyme cleavage kinetics: A circular DNAzyme (C-Dz) containing a nonfunctional region and a linear DNAzyme (L-Dz) were analyzed for cleavage kinetics. A substrate reporter concentration gradient was used: 0.156, 0.312, 0.625, 1.25, 2.5, and 5 μM. The total reaction volume was 100 μL, and the buffer consisted of 50 mM NaCl, 10 mM Tris-HCl, and 1 mM MgSO₄. Reaction progress was monitored at 37°C using a Thermo Scientific VariOskan Flash spectrometer (ThermoFisher, Waltham, MA, USA). Fluorescence signals were recorded every 120 seconds for a total of 15 measurements. Fluorescence values ​​were converted to substrate cleavage concentrations (nM) using a fluorescence calibration curve. Kinetic parameters were calculated using the Michaelis–Menten equation, using the initial reaction rate (nM s⁻¹) calculated from the first 120 seconds of the linear segment of the reaction curve. The reaction rate-substrate concentration data were fitted to the Michaelis-Menten equation using GraphPad Prism 9 software (GraphPad, California, USA) to obtain the catalytic constant k. cat and Michaelis constant K M The experimental results were verified by 12% polyacrylamide gel electrophoresis (PAGE).

[0045] Molecular dynamics simulation: In order to explore the differences in DNA-RNA binding between different structural systems under aqueous solvent conditions, molecular dynamics simulations were performed using GROMACS2021.5 software. All systems were established in a closed environment with a temperature of 300K and a pressure of 1 bar. Periodic boundary conditions were set using the GROMACS software so that the closest distance between the edge of the molecular complex and the boundary of the simulation box was 1.0 nm. The receptor topology file was converted to a GROMACS-recognizable format using the pdb2gmx tool, and the parameter set was AMBeff14SB. AmberTools was used to generate GROMACS-compatible topology files for small molecules, the ligand atoms were processed using the GAFF force field, and the water molecule model was modeled using TIP3P. After the system was initialized, all atoms were energy minimized using the steepest descent method. After the simulation, the GROMACS module was used to calculate the root mean square deviation (RMSD) and root mean square fluctuation (RMSF), and the molecular force field / generalized Born surface area method (MM / GBSA) was used to calculate the ligand binding free energy (ΔG of binding).

[0046] Real-time monitoring and characterization of C-Dz-mediated RCA reactions: A 20 μL rolling circle amplification (RCA) reaction system consisted of 1× phi29 DNA polymerase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 10 mM (NH₄)₂SO₄, 4 mM DTT), 1× T4 polynucleotide kinase (PNK) buffer (70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT), 2 μL of circular probe, 2 μL of target RNA (1 μM), 2 μL of hairpin probe (300 nM), 2 μL of dNTPs (10 mM), 0.5 μL of phi29 DNA polymerase, 0.5 μL of T4PNK, 0.2 μL of recombinant albumin, 0.2 μL of recombinant RNase inhibitor, and 1.2 μL of MgSO₄. Fluorescence signals were recorded every 15 seconds at 37°C using the FAM channel of a BioRad CFX 96 real-time PCR instrument (USA). The raw fluorescence curve was fitted with the cumulative distribution function (CDF) of the gamma distribution using custom MATLAB code to obtain the α and β parameters for calculating the theoretical fluorescence peak time. TIME-FLUO strategy: In addition to the above steps, fluorescence signals were recorded simultaneously in the FAM, HEX, Texas Red, and Cy5 channels with a time interval of 15 seconds.

[0047] Construction of the CLOCK Multiplex Time Barcode Diagnostic Platform: The C-DzTIME block design was optimized for multiple respiratory virus detection. 2 μL of circular probe for each target RNA was mixed with 2 μL of target RNA (1 μM) in an optimized RCA reaction system, and FAM fluorescence was immediately monitored. Reaction curves were analyzed using custom MATLAB code, and the effectiveness of the synthesis of C-Dz molecules of varying lengths was verified by 12% PAGE.

[0048] Pseudovirus Sample Validation: Real-time RT-PCR was used as the gold standard to evaluate the consistency of the CLOCK method for the detection of four pseudoviruses: influenza A (H1N1), influenza B (FLUB), respiratory syncytial virus (RSV), and human rhinovirus (HRV). Viral RNA was extracted using the QIAamp Viral RNA Mini Extraction Kit protocol, and concentration was determined using a NanoDropOne spectrophotometer (Thermo Fisher, USA). cDNA synthesis was performed using the AMV First-Strand cDNA Synthesis Kit. A 20 μL reverse transcription system containing 5 μL total RNA, 1 μL oligonucleotide primer, and 5 μL RNase-free water was pre-denatured at 65°C for 5 minutes and then incubated on ice for 30 seconds. Subsequently, 4 μL 5× reaction buffer, 1 μL RNase inhibitor (40 U / μL), 2 μL dNTPs (10 mM), and 2 μL AMV reverse transcriptase (10 U / μL) were added. The reaction was incubated at 42°C for 60 minutes and inactivated at 85°C for 5 minutes. Real-time quantitative PCR: A 20 μL reaction system containing 2 μL DNF buffer, 10 μL 2×SG fast qPCR master mix, 0.4 μL forward and reverse primers (10 μM), 6 μL cDNA, and 1.2 μL water was mixed on ice. The reaction procedure included preactivation at 95°C for 5 minutes; denaturation at 95°C for 3 seconds followed by annealing and extension at 60°C for 30 seconds for 50 cycles; and melting curve analysis (65°C to 95°C, 0.5°C / step).

[0049] Clinical Sample Testing: 300 nasopharyngeal swab samples (160 uninfected, 100 infected with influenza A, 20 infected with RSV, and 20 infected with rhinovirus) were collected. All samples were clinically confirmed by RT-PCR at the Southwest Hospital of the Army Medical University (Chongqing, China) between November and December 2024. Viral RNA was extracted and purified using the RNeasy mini kit and stored at −80°C until use. This study was approved by the Ethics Committee of the First Affiliated Hospital of the Army Medical University and adhered to the Declaration of Helsinki and the International Ethical Guidelines for Biomedical Research Involving Humans.

[0050] The sequences involved in this application are shown in Table 1.

[0051] Table 1 Nucleotide sequences involved in this application

[0052]

[0053]

[0054]

[0055]

[0056] Example 1 Overview of the invention: Design and construction of a closed circular DNA enzyme kit with multi-target diagnostic function ( clo sed C -Dz k it, referred to as CLOCK)

[0057] The present invention designs a functional region in a linear DNAzyme (L-Dz), and then cyclizes the L-Dz by adjusting the topological structure to obtain an engineered circular DNAzyme (C-Dz). The circular DNA has excellent rigidity and thermodynamic stability. The functional region is divided into a hairpin-encoded region for activating a fluorescent signal (Fluorescence, abbreviated as FLUO) and a temporal coding region (Temporal dentified region) composed of repeated T sequences. Me The fluorescent coding region has a sequence complementary to the hairpin probe carrying the fluorescent group, while the time coding region is a repeated thymine (T) sequence to introduce a time delay. The time coding region is located upstream of the signal output region. The engineered C-Dz retains the catalytic core region (Catalytic core) of RNA cleavage function and also contains detection arms (Detection arms) with sequence-specific recognition capabilities. The preparation principle diagram is shown in FIG. Figure 1 As shown in A.

[0058] After the engineered C-Dz recognizes the target RNA, its detection arms specifically bind to the target, and the catalytic core region is located in the Mg 2+In the presence of a molecule, the target is catalyzed and cleaved, generating a new 3' end on the RNA and triggering rolling circle amplification (RCA). The fluorescent coding region in the engineered C-Dz is paired with a hairpin probe carrying a fluorescent group in the reaction system. The hairpin structure opens to restore fluorescence, generating a two-dimensional code containing a fluorescent signal and a time signal. In the engineered C-Dz, since the length of the TIME region can be changed by editing, after several rounds of RCA reactions, the peak time of the fluorescence intensity of the engineered C-Dz and the original encoded C-Dz will have a significant time difference (TIME to Fluorescent Peak Difference), such as Figure 1 As shown in B.

[0059] By utilizing this characteristic, different types of engineered C-Dz with different fluorescence coding regions and time coding regions of different lengths can generate different dynamic TIME-FLUO two-dimensional barcodes through RCA reaction. This method expands the existing fluorescent barcode technology into a two-dimensional coding system, integrating dynamic time signals and fluorescence signals to achieve multi-target analysis ( Figure 1 C).

[0060] The present invention uses the above principle to construct the CLOCK system, and clinically applies it to the simultaneous detection of four respiratory viruses, such as Figure 1 As shown in Figure 2, when the CLOCK system is used to detect multiple target RNAs, CLOCK generates specific TIME-FLUO 2D barcodes, thereby identifying multiple targets that can be decoded from the signal.

[0061] Example 2: Differences in the cleavage of C-Dz and L-Dz

[0062] In this example, the structural stability of C-Dz and L-Dz in high concentration solutions of Exonuclease I and Exonuclease III was compared. The sequences of C-Dz, L-Dz, reporter probe substrate, and C-Dz circular primer are shown in SEQ ID NOs. 30 to 33. The reaction system consisted of a reaction buffer solution containing 50 mM NaCl, 10 mM Tris-HCl, and 1 mM MgSO4. The two groups of DNA enzymes were reacted with 40 U of Exonuclease I (Exo I) and 100 U of Exonuclease III (Exo III), respectively, at 37°C for 30 minutes, and then subjected to 12% PAGE for 50 minutes. The results are shown in FIG. Figure 2 As shown in A, C-Dz with a closed-loop structure can maintain its integrity and is extremely resistant to exonuclease digestion for 2 hours or longer, while L-Dz is almost completely digested within 15 minutes. Therefore, the biological stability of C-Dz provides the basis for its cleavage efficiency.

[0063] Then C-Dz and L-Dz were subjected to DNA enzyme rolling circle amplification (RCA) experiment to compare their cleavage activities. Figure 2 As shown in B (taking C-Dz as an example), inactive DNA enzyme is in the presence of magnesium ions (Mg 2+ ) is converted into active DNA enzyme in the presence of , cleaving FAM-BHQ1 target RNA, generating fluorescent signals, allowing the active DNA enzyme to release the target RNA and proceed to the next cycle; 12% PAGE is used to analyze the Figure 2 The final product in B and the control group were analyzed by gel electrophoresis. Figure 2 As shown in Figure C, lane M: 20-bp DNA marker; lane 1: only substrate RNA (1M) in the DNA enzyme cycle; lane 2: only L-Dz (1M) in the DNA enzyme cycle; lanes 3 and 4: substrate RNA (1M) and L-Dz (1M); lane 5: substrate RNA (1μM) and C-Dz (1μM). It can be seen from the figure that the efficiency of C-Dz in cleaving RNA is almost equivalent to that of linear L-Dz.

[0064] The kinetics of C-Dz and L-Dz were quantitatively compared. The concentration gradient of the substrate was 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, 2.5 μM and 5 μM. The fluorescence was recorded every 120 s to obtain the relationship between fluorescence intensity and reaction time, as shown in Figure 2. Figure 2 As shown in D; Figure 2 E is the linear relationship between fluorescence intensity and substrate concentration. It can be seen from the figure that fluorescence intensity and substrate concentration show a good linear regression (R 2 >0.95), so there is no fluorescence inner filter effect in this system.

[0065] The mechanism of the difference in substrate cleavage performance between C-Dz and L-Dz was studied. In this example, the functional region lengths of C-Dz and L-Dz were set to 45 nt, and molecular dynamics simulations were performed. The sequences of C-Dz, L-Dz, and RNA are shown in SEQ ID NOs. 34 to 36. The experiment found that within a simulation time of 100 ns, compared with L-Dz / RNA, the C-Dz / RNA model reacted to a rigid triangular structure, with a shorter and more stable cleavage site base distance and higher catalytic efficiency. In the L-Dz / RNA model, due to the shorter and longer cleavage site distances, the linear ends were oscillating, thereby inhibiting the catalytic efficiency ( Figure 2 F). This simulation demonstrates the better thermodynamic stability and higher cleavage activity of C-Dz. The root mean square deviation (RMSD) of DNA shows that the L-Dz / RNA model is slightly higher than the C-Dz / RNA model, indicating that the swings at both ends of C-Dz are smaller than those of L-Dz ( Figure 2G). Similarly, the RMSD of the two DNA enzyme RNAs after stabilization did not differ significantly, and throughout the simulation, the C-Dz / RNA model reached structural equilibrium faster than the L-Dz / RNA model, suggesting that the structural angle of the closed-ring C-Dz itself produced a faster conformational transition during the binding of C-Dz to the target RNA, improving the catalytic efficiency ( Figure 2 H). The root mean square fluctuation (RMSF) of the middle part of DNA is not much different, but the root mean square fluctuation of the nucleotides at both ends of L-Dz / RNA is higher, which confirms that it has greater oscillation than C-Dz / RNA, as shown by RMSD ( Figure 2 I). The RMSF of RNA 3'-end is not much different ( Figure 2 J). In the L-Dz / RNA model, the 3'-terminal RNA exhibits RNA-RNA pairing, whereas in the C-Dz / RNA model, these RNA moieties pair with C-Dz, suggesting that C-Dz restricts the pairing space of target molecules to a greater extent than L-Dz. Radial distribution function (RDF) plots show that the C-Dz / RNA system is more compact than the L-Dz / RNA system, with a shorter distance between cleavage sites, thereby enhancing catalytic ability ( Figure 2 K). The binding free energy of C-Dz / RNA is significantly lower than that of L-Dz / RNA, indicating that C-Dz binds tightly and stably to the target RNA ( Figure 2 L).

[0066] According to the above analysis, compared with L-Dz, the closed-ring structure of C-Dz has faster structural conversion, more stable dynamics, shorter cleavage site distance, and tighter binding reaction, thereby improving the catalytic efficiency.

[0067] Example 3 Preparation of Engineered C-Dz

[0068] In this embodiment, four time coding regions of different lengths were constructed, which were named TIME-10 (10 nt), TIME-20 (20 nt), TIME-30 (30 nt) and TIME-40 (40 nt). The sequences of C-Dz of TIME-10, TIME-20, TIME-30 and TIME-40 were shown in SEQ ID NO.21 to SEQ ID NO.24, respectively. The looping primer was shown in SEQ ID NO.25. Figure 3 As shown in A, the feasibility of time coding regions of different lengths for delaying the fluorescence peak time was verified; the real-time fluorescence curves of four groups of amplifications were recorded, and the differences between different time coding regions and the fluorescence peak were shown in Figure 3 As shown in Figure B, it can be seen that the longer the time coding region is, the later the peak time of fluorescence intensity is; Figure 3C shows the SDS-polyacrylamide gel electrophoresis analysis of C-Dz and L-Dz in each time coding region, indicating that C-Dz with four different lengths of time coding regions inserted were successfully prepared; Figure 3 D is the gamma-fitted probability distribution function (PDF) of the four fluorescence curves generated by the four engineered C-Dz calculated by MATLAB code. By calculating the derivative of the change in fluorescence intensity over time, a curve of the instantaneous intensity change over time is obtained, where the shape parameter (α) and scale parameter (β) are calculated by MATLAB to determine the theoretical peak time ΔT of the initial original fluorescence curve; and the peak fluorescence time of the four engineered C-Dz is as follows Figure 3 As shown in E, there is statistical significance between the peak time values, and asterisks indicate the statistical significance level: P < 0.0001 (****), P < 0.001 (***), P < 0.01 (**).

[0069] Based on the above results, it was found that engineered C-Dz inserted into time coding regions of different lengths could amplify the cumulative time difference through RCA, thus verifying that the construction of TIME barcodes is feasible, e.g. Figure 3 As shown in F.

[0070] Furthermore, this embodiment combines a fluorescence coding region with a time coding region to construct a two-dimensional TIME-FLUO dynamic barcode. An engineered C-Dz containing both a fluorescence coding region and a time coding region is constructed. The fluorescence coding region is a complementary sequence to a hairpin probe. Through RCA cycles, the engineered C-Dz binds to the hairpin probe, and the hairpin opens, restoring fluorescence. Therefore, M types of fluorescence can encode M types of fluorescence barcodes, and N types of time coding regions can encode N types of time barcodes. Combining M types of fluorescence barcodes with N types of time barcodes enables high-throughput TIME-FLUO (N×M types) target decoding.

[0071] This example introduces four fluorescent groups: FAM (6-Carboxyfluorescein), HEX (Hexachlorofluorescein), Texas Red (Sulforhodamine 101sulfonylchloride), and Cy5 (Cyanine 5) (the corresponding hairpin probe sequences are shown in SEQ ID NO. 26 to SEQ ID NO. 29). Their fluorescence spectra do not overlap with each other, as shown in FIG. Figure 3 As shown in Figure G, fluorescence analysis showed no statistically significant difference in the time to peak fluorescence intensity for time-coded regions of the same length. Therefore, two-dimensional TIME-FLUO barcodes have broad application prospects in multi-target analysis.

[0072] Example 4 Detection of the effect of single arm length on DNAzyme cleavage activity

[0073] Take the process of circular DNAzyme amplification as an example, Figure 4 As shown in A, the circular DNAzyme includes the DNA enzyme catalytic core, the detection region, and the functional region. The inactive DNAzyme is activated by magnesium ions (Mg 2+ ) is converted into an active DNAzyme in the presence of , and cleaves the reporter substrate, one end of which is connected to a FAM fluorescent group and the other end is connected to a quenching group BHQ1 (Black Hole Quencher-1). After the reporter RNA is cleaved, a fluorescent signal is generated, causing the active DNA enzyme to release the target RNA for subsequent cycles.

[0074] In this example, six linear DNAzymes and six circular DNAzymes were prepared according to the preparation method. The lengths of their detection arms were 5 nt, 8 nt, 10 nt, 12 nt, 15 nt, and 20 nt, respectively. The DNAzymes with different detection arms were verified by 12% PAGE SDS-polyacrylamide gel electrophoresis. The verification results are shown in FIG. Figure 4 As shown in B, it indicates that the preparation is successful; the sequences of the six linear DNAzymes, six circular DNAzymes, and their reporter probes are shown in SEQ ID NO.38 to SEQ ID NO.55, and the circular primer of the circular DNAzyme is shown in SEQ ID NO.37.

[0075] The prepared DNAzyme was subjected to substrate cleavage. The experimental steps were as described above. The real-time fluorescence curves under gradient substrate concentrations were as follows. Figure 4 As shown in Figure C, the substrate concentration gradient was 0.078 μM, 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, and 2.5 μM, respectively. All fluorescence data were recorded using a ThermoScientific VariOskan Flash multifunctional microplate reader (Massachusetts, USA). Data are expressed as mean ± SD (n = 3 independent experiments).

[0076] Then, the cleavage kinetics of six L-Dz and six C-Dz were tested. In this example, the fluorescence signals of each were converted into Michaelis-Menten curves generated by the cleavage substrate concentration to calculate k cat and K M The kinetic test results are as follows Figure 5 As shown in A, for 5nt: k of C-Dz and L-Dzcat and K M 1.55×10 7 and 7.98×10 6 , maximum V C-(Dz) / V L-(Dz) The ratio is about 3; for 8 nt: k of C-Dz and L-Dz cat and K M They are 2.91×10 8 and 5.06×10 7 , maximum V C-(Dz) / V L-(Dz) Ratio <7; for 10 nt: k of C-Dz and L-Dz cat and K M They are 1.12×10 8 and 3.22×10 7 , maximum V C-(Dz) / V L-(Dz) The ratio is about 4; for 12nt: k cat and K M 5.98×10 7 and 2.66×10 7 , maximum V C-(Dz) / V L-(Dz) The ratio is about 3; for 15nt: k of C-Dz and L-Dz cat and K M They are 4.48×10 7 and 1.94×10 7 , maximum V C-(Dz) / V L-(Dz) The ratio is close to 2; for 20nt: k cat and K M They are 4.71×10 7 and 2.31×10 7 , maximum V C-(Dz) / V L-(Dz) The ratio was close to 2, mean ± SD, n = 3. The above results showed that regardless of the length of the single arm tested, the catalytic efficiency of C-Dz was higher than that of L-Dz, which confirmed the excellent target cleavage efficiency of C-Dz. In particular, 8nt C-Dz achieved a balance between complementary pairing strength and conformational flexibility. The catalytic efficiency of 8nt C-Dz was nearly 7 times that of 8nt L-Dz.

[0077] The cleavage rates of different length detection arms (Single arm detection region) and initial substrate concentrations (Initial substrate concentration) are summarized as follows: Figure 5As shown in B, under the same substrate concentration, the cleavage rate was highest when the single arm length was 8 nt.

[0078] Example 5 Effect of functional region length on DNAzyme cleavage activity

[0079] In this example, four linear DNAzymes and four circular DNAzymes were prepared according to the preparation method. The functional region lengths of these four linear DNAzymes, four circular DNAzymes, and their reporter probes were 15 nt, 35 nt, 50 nt, and 65 nt, respectively. The sequences of the four linear DNAzymes, four circular DNAzymes, and their reporter probes are shown in SEQ ID NO. 57 to SEQ ID NO. 65. The circular primers of the circular DNAzymes are shown in SEQ ID NO. 56.

[0080] The cleavage kinetics experiments were performed for all DNAzymes. In this example, the fluorescence signals of each DNAzyme were converted into Michaelis-Menten curves generated by the cleavage substrate concentration to calculate the k cat and K M The kinetic test results are as follows Figure 6 As shown in A, for 15nt: k of C-Dz and L-Dz cat and K M 1.725×10 7 and 6.411×10 7 , maximum V C-(Dz) / V L-(Dz) Ratio > 2; for 35nt: k of C-Dz and L-Dz cat and K M are 1.510×10 7 and 6.413×10 7 , maximum V C-(Dz) / V L-(Dz) Ratio > 2; for 50 nt: k of C-Dz and L-Dz cat and K M are 1.744×10 7 and 6.96×10 7 , maximum V C-(Dz) / V L-(Dz) The ratio is about 3; for 65nt: k of C-Dz and L-Dz cat and K M are 1.577×10 7 and 7.265×10 7 , maximum V C-(Dz) / V L-(Dz) The ratio is about 3.

[0081] The cleavage rates of functional regions of different lengths and initial substrate concentrations are summarized as follows: Figure 6 As shown in B.

[0082] Example 6 Construction of CLOCK for Multiple Time Barcode Detection of Respiratory Viruses clo sed C -Dz k it)

[0083] Based on the above experiments, this embodiment constructs four time coding regions, corresponding to four respiratory viruses, such as Figure 7 As shown in A, H1N1 RNA (influenza A (H1N1) virus RNA) corresponds to the 15nt temporal coding region, FLUB RNA (Influenza B virus RNA) corresponds to the 30nt temporal coding region, RSV RNA (Respiratory Syncytial Virus RNA) corresponds to the 45nt temporal coding region, and HRV RNA (Human rhinovirus RNA) corresponds to the 65nt temporal coding region (the corresponding C-Dz sequences are shown in SEQ ID NO.1 to SEQ ID NO.4, the hairpin probe sequences are shown in SEQ ID NO.9 to SEQ ID NO.12, and the looping primer is shown in SEQ ID NO.25).

[0084] After rolling circle amplification of the engineered C-Dz, there were significant differences in the corresponding fluorescence peak times, such as Figure 7 As shown in B, asterisks indicate statistical significance levels, p < 0.001 (***), p < 0.01 (**); gel electrophoresis verification showed that C-Dz and L-Dz inserted into different time coding regions were successfully prepared, see Figure 7 C.

[0085] Next, this example uses four pseudoviruses (H1N1, FLUB, RSV, and HRV, whose sequences are shown in SEQ ID NO.5 to SEQ ID NO.8) as samples for verification. According to the experimental results, each target RNA corresponding to H1N1, FLUB, RSV, and HRV showed ultra-high detection sensitivity, with a detection value as low as 1 fM and a linear regression R 2 The values ​​are 0.9468, 0.9623, 0.9746 and 0.9420 respectively. Figure 7 D; the CLOCK platform was compared with the gold standard - reverse transcription polymerase chain reaction (RT-PCR), and it was found that the two showed good consistency, as shown in Figure 7E shows, R 2 They are 0.9606, 0.9517, 0.9712 and 0.9919 respectively.

[0086] In order to demonstrate the feasibility of the CLOCK platform of the present invention in practical applications, clinical samples (non-simulated samples) were collected using nasopharyngeal swabs, and target RNA sequences were extracted. Two-dimensional barcodes were generated using CLOCK, as shown in Figure 2. Figure 7 F; All 300 samples were clinically validated by RT-qPCR, including 100 H1N1 positive samples, 100 H1N1 negative samples, 20 FLUB negative samples, 20 RSV positive samples, 20 RSV negative samples, 20 HRV positive samples and 20 HRV negative samples; All 300 swab samples were tested using the CLOCK platform, and significant differences were found between positive and negative samples (p < 0.0001) ( Figure 7 G); CLOCK was compared with the clinical validation results of RT-qPCR (the primers required for RT-qPCR are shown in SEQ ID NO. 13 to SEQ ID NO. 20). We determined that the clinical sensitivity and specificity of CLOCK for H1N1 were 100% and 99%, respectively, for RSV were 100% and 85%, respectively, for HRV were 100% and 85%, and for FLUB was 100%, indicating that CLOCK performed well in clinical detection, such as Figure 7 As shown in H.

[0087] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for constructing an engineered C-Dz, characterized in that: A fluorescence coding region and a time coding region are inserted into L-Dz to obtain engineered L-Dz, the length of the L-Dz detection single arm is set to 5-12 nt, and the engineered L-Dz is cyclized to obtain engineered C-Dz.

2. The construction method according to claim 1, characterized in that The length of the L-Dz detection single arm is 8 nt.

3. The construction method according to claim 1, characterized in that The time coding region is arranged downstream of the engineered C-Dz recognition region, and the fluorescence coding region is arranged downstream of the time coding region.

4. The construction method according to claim 1, characterized in that The temporal coding region is a repeated thymine sequence.

5. The engineered C-Dz constructed according to the construction method according to any one of claims 1 to 4.

6. A method for constructing a TIME-FLUO barcode, characterized in that: The engineered C-Dz described in claim 5 generates a two-dimensional TIME-FLUO barcode containing time and fluorescence signals after rolling circle amplification.

7. The construction method according to claim 6, characterized in that: The engineered C-Dz comprises at least two engineered C-Dz with different time coding region lengths.

8. The construction method according to claim 7, characterized in that: The spectra of the fluorescent groups of the hairpin probes matched with the fluorescent coding regions corresponding to the engineered C-Dz with different time coding region lengths do not overlap with each other, and the time coding regions with different lengths differ by 10-20 nt.

9. Use of the TIME-FLUO barcode constructed according to the construction method of claim 7 or 8 in preparing a multiple virus detection kit.

10. The use according to claim 9, characterized in that The multiple virus detection kit is used to detect respiratory viruses; the respiratory viruses are a combination of one or more of H1N1, FLUB, RSV and HRV.

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