Method for improving nucleic acid connection fidelity based on selenium-modified ATP (adenosine triphosphate) and application

Abstract

The invention belongs to the technical field of biology, in particular to the technical field of biomedicine, and particularly relates to a method for improving fidelity of DNA and RNA and DNA-RNA mixed connection based on selenium modified ATP and application, ATP alpha Se is used for enhancing the mismatch recognition capacity of DNA ligase and RNA ligase, and ATP alpha Se can improve the connection accuracy of multiple ligase. According to the present invention, ATP [alpha] Se is used, the key selective interaction of the ligase active site is specifically improved through the selenium atom, the ligase is sensitive to the substrate mismatch of the large expansion region, and the fidelity of the four-base protruding terminal connection in almost all the connection sequences is improved by using the ATP [alpha] Se technology; the ATP alpha Se is used, so that the accuracy of Golden Gate Assay is improved, the accuracy of simultaneous assembly of multiple fragments is improved, the success rate is improved, and the cost of genome assembly can be reduced; compared with the prior art, the kit has the advantages that the kit has the advantages that the kit can be used for detecting multiple SNP genes, false positive in multiple ligation-dependent probe amplification (MLPA) reaction can be effectively inhibited, the accuracy of gene detection with multiple SNPs at the same time can be higher, and the cost of simultaneous detection of multiple genes can be reduced.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology, biomedical technology, and nucleic acid (DNA and RNA) ligation technology, and particularly relates to a method and application for improving the accuracy and fidelity of nucleic acid ligation based on selenium-modified ATP. Background Technology

[0002] Nucleic acid-associated enzymes (NAIs) are crucial for the accurate replication, transcription, and translation of nucleic acids, enabling a wide range of biotechnological applications, including gene synthesis, gene editing, nucleic acid detection, and the preparation of modified mRNA. DNA ligases and RNA ligases play a vital role in this process. DNA or RNA ligases are activated by ATP (they are called cofactors or substrates) and, in the presence of a complementary nucleic acid ligation template, catalyze the formation of a phosphodiester bond between the 3'-hydroxyl group (3'-OH) of the recipient DNA (or RNA) and the 5'-terminal phosphate group (5'-P) of the donor DNA (or RNA). While ligases can identify base mismatches between the ligation template and the substrate to some extent, this identification is insufficient for most applications.

[0003] For example, mismatches in substrate DNA can distort the geometry of the active site, reducing catalytic activity and thus enabling mismatch identification. However, the mismatch-gap ligation resolution of DNA ligases is limited to base pair mismatches (one or two nucleotides) adjacent to the gap, and the resolution of donor 5'-P-terminal mismatches is often lower than that of acceptor 3'-OH-terminal mismatches. These limitations pose challenges to ligase-based applications such as nucleic acid detection and gene synthesis. For instance, Golden Gate Assembly, a key technique in genome synthesis, uses IIS-type restriction enzymes to create unique 5'-hangovers (4nt) on DNA fragments, which are then ligated together in a one-pot reaction using end-ligating DNA ligases (typically T4 DNA ligases). However, the poor discrimination ability of DNA ligases hinders high-fidelity ligation, especially in ligations with 5'-hangovers and those containing numerous hangover sequences, thus impeding the efficient assembly of multiple fragments in a single reaction.

[0004] T4 DNA ligase is a commonly used enzyme in DNA ligation reactions, and it can also be used for RNA and DNA-RNA mixed ligation. It is widely used in gene cloning, recombinant DNA technology, and nucleic acid detection. This is because T4 DNA ligase not only has high activity and good stability, but it can also recognize various substrate types, including nicked double-stranded DNA (nDNA), sticky-end DNA, and blunt-end DNA. However, this enzyme has a high tolerance for mismatches, which can easily lead to non-specific ligation and affect the accuracy of experimental results, especially in applications requiring high-precision ligation. Although many high-fidelity ligases have been discovered and identified to improve the accuracy of nucleic acid ligation, their activity is very low in the ligation of sticky-end DNA substrates, making them unsuitable for effective application, such as in genome assembly. Therefore, improving the ligation fidelity of T4 DNA ligase and enhancing its accuracy in DNA ligation reactions is of great significance for scientific research and biotechnology applications.

[0005] While existing methods for improving the fidelity of nucleic acid ligation are effective, they also have certain drawbacks and limitations. The specific methods for improving the fidelity of nucleic acid ligation include the following:

[0006] 1) As analyzed above, high-fidelity ligases such as Taq DNA ligase and 9°N DNA ligase are used. However, high-fidelity ligases usually have low activity for sticky end ligation, which limits their applicability.

[0007] 2) Optimize reaction conditions: Adjust factors such as ATP concentration, magnesium ion concentration, and salt concentration in the reaction system to improve the fidelity of the ligation reaction. For example, reducing the ATP concentration can reduce the occurrence of mismatches; however, optimizing reaction conditions is complex: too low an ATP concentration will reduce ligation activity and decrease ligation yield.

[0008] 3) Use of additives: Adding specific chemicals, such as dimethyl sulfoxide (DMSO) or urea, to the ligation reaction can increase substrate annealing specificity, reduce mismatches, and thus improve ligation fidelity. However, the use of additives still carries risks: inappropriate additives or concentrations may inhibit enzyme activity, affecting ligation efficiency.

[0009] These methods typically offer limited improvement in ligation fidelity in key bioengineering reactions catalyzed by ligases; they also suffer from poor versatility, requiring optimization for each ligation system. Furthermore, Golden Gate assembly generally requires the generation of relatively short paired ends via endonucleases, followed by the use of natural ATP as a cofactor to ligate the DNA substrate. However, due to the current low ligation fidelity, multiple batch assemblies are often necessary when ligating multiple fragments, increasing the cost and time of genome assembly. In addition, MLPA (Multiplex Ligation Dependent Probe Amplification) SNP (single nucleotide polymorphism) detection protocols usually require the target and probe to be paired and ligated, followed by PCR amplification of the ligated probe to generate a signal. However, current detection methods may produce false positives due to low SNP site recognition, potentially preventing the simultaneous detection of different types of single-base differences in a single batch.

[0010] In summary, although existing methods have made some progress in improving the fidelity of nucleic acid ligation, challenges remain in terms of efficiency, stability, and applicability.

[0011] ATPαSe can act as a cofactor for ligases in DNA / RNA ligation, and can also provide energy for many other reactions (see ATP).

[0012] Chinese invention patent CN202080044433.0 describes modified nucleotides and methods for DNA and RNA polymerization and sequencing. Modified nucleotides, such as α-phosphoselenonucleotides (dNTPαSe and NTPαSe), can be incorporated into nucleic acids via enzymatic methods in a manner similar to that of natural nucleotides. Changing the properties of the modified nucleotides can alter the interaction between the nucleotides and enzymes. Although the enzymatic incorporation rate of modified nucleotides may be slightly lower than that of natural nucleotides, it can significantly suppress the erroneous incorporation of nucleotides into nucleic acids during enzymatic extension and / or polymerization methods. In this patent, dNTPαSe and NTPαSe are used as polymerase substrates. Summary of the Invention

[0013] To address the above technical problems, this invention provides a method and technique for using selenium-modified ATPαSe cofactors to improve the fidelity of DNA and RNA ligation based on ATP cofactors. The application of this method can significantly identify the base pairing of substrates, inhibit the ligation of mismatched substrates, and relatively increase the ligation efficiency of right-matched substrates; thereby enabling DNA and RNA ligation to have high fidelity, stability, and applicability.

[0014] The present invention, which addresses the above-mentioned technical problems, describes the use of selenium-modified ATPαSe cofactor. ATPαSe replaces natural ATP as a ligase cofactor to enhance the mismatch recognition ability of ligases in DNA and RNA ligation, helping ligases identify correct base pairs. This improves the fidelity of DNA and RNA ligases and further enhances the accuracy of DNA ligases in multi-SNP nucleic acid detection, seamless cloning in Kinmen, or multiplex ligation-dependent probe amplification (MLPA).

[0015] In the optimized scheme, the DNA ligase is T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, or other DNA ligases that use ATP as a cofactor.

[0016] In the optimized scheme, the RNA ligase is T4 RNA ligase 2 or other RNA ligases with ATP as a cofactor.

[0017] The molar dosage of the ATPαSe cofactor is 1 μM-50 mM.

[0018] Furthermore, the molar amount of the ATPαSe cofactor is 0.2 mM.

[0019] The DNA ligation system has four main components: enzyme, substrate, buffer (ion), and cofactor. Conventionally, improving fidelity is achieved by altering the first three components. This invention uses ATPαSe as a cofactor for the ligase to improve the fidelity of DNA ligation.

[0020] The application of the ATPαSe cofactor in the preparation of reagents for multiple ligation-dependent probe amplification detection.

[0021] The application of the ATPαSe cofactor in the preparation of reagents for assembling seamless clones in Kinmen.

[0022] The ATPαSe refers to the product in which the α-phosphorus non-bridging oxygen atom of ATP is replaced by a selenium atom, and it contains two diastereomers. The present invention uses the diastereomer (ATPαSe Peak1) which migrates faster in reverse HPLC.

[0023] The present invention discloses a method for improving DNA ligation fidelity based on selenium-modified ATP, which includes applying the ATPαSe cofactor as described above to the DNA ligation reaction system to enhance the mismatch recognition ability of DNA ligase by utilizing ATPαSe; or its application in the assembly of seamless clones in Kinmen, or its application in multiple ligation-dependent probe amplification detection.

[0024] In the optimized scheme, the DNA-RNA ligation reaction system includes the ATPαSe cofactor.

[0025] Specifically, the mixture for the ligation reaction, i.e. the DNA ligation reaction system, consists of 0.2 mM ATPαSe, 1× ligase buffer (40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM DTT), T4 DNA ligase (final concentration 50 nM to 400 nM), and double-stranded DNA substrate (final concentration 200 nM to 1000 nM).

[0026] Furthermore, the reaction time is 1 min to 48 h and the temperature is 4 °C to 45 °C.

[0027] The principle behind this invention, which utilizes ATPαSe to enhance the mismatch recognition ability of DNA ligase, is as follows:

[0028] By replacing the non-bridging O atom connected to the P atom at the α-position of ATP with a Se atom, the interaction between ATP and specific atoms of the ligase is weakened, particularly the stability of the complex formed between the mismatch substrate and the ligase. This increases the difference in ligase activity between positive and mismatch substrates, thus enhancing the mismatch recognition ability of DNA ligase using selenium-modified ATPαSe. Other accuracy improvements in applications are based on this enhanced mismatch recognition ability of DNA ligase.

[0029] Since AMP-activated intermediates play a crucial role in maintaining active conformations (including conformations in mismatch ligation), this invention also provides a method for improving DNA ligation fidelity based on cofactors. This method includes applying the aforementioned ATPαSe cofactor to the DNA ligation reaction system, utilizing ATPαSe to enhance the mismatch recognition ability of DNA ligase, and its application in Golden Gate assembly of seamless clones or in multiplex ligation-dependent probe amplification (MLPA). Adding ATPαSe to the ligation reaction system improves ligation accuracy, and selenium-modified ATP enhances DNA ligation fidelity.

[0030] Effects of this invention:

[0031] (1) ATPαSe replaces natural ATP as a cofactor for ligases. It is used in DNA and RNA ligation based on nucleic acid ligation templates to enhance the ability of DNA ligase and RNA ligase to distinguish base mismatches between the ligation template and the ligation substrate, and to help improve the ability of DNA ligase and RNA ligase to recognize base correct matches, thereby improving the accuracy of DNA ligase and RNA ligase.

[0032] (2) The use of ATPαSe enhances the ligase’s ability to identify mismatches over a wider range.

[0033] (3) The use of ATPαSe increases the fidelity of DNA ligation with most of the sticky end sequences.

[0034] (4) The use of ATPαSe improved the accuracy of one-pot assembly of multiple gene fragments based on the Golden Gate protocol, and the positive rate of assembly in 15 fragments was more than 200 times higher than that of ATP-based assembly.

[0035] (5) Using ATPαSe can improve the specificity of multiple linkage-dependent probe amplification (MLPA) technology and suppress false positive signals that occur in the simultaneous detection of multiple SNPs based on MLPA technology. Attached Figure Description

[0036] Figure 1 This is a characterization of the synthesis of ATPαSe in this invention.

[0037] (Among them, A. the molecular structure of ATPαSe and the relative retention times of the two diastereomers in reverse HPLC; B. the specific interaction between the substrate nucleic acid and the ligase caused by selenium atom substitution for oxygen atom (simulation of selenium atom substitution based on the known complex structure (PDBID: 6DT1)); C. a schematic diagram of how ATPαSe inhibits mismatch ligation and improves ligation accuracy.)

[0038] Figure 2 This serves as a verification of the universality of the ATPαSe method used in this invention to enhance the fidelity of DNA ligase.

[0039] (Among them, A. the ligation of T3 DNA ligase using ATP or ATPαSe as cofactors at +1 and -1 sites; B. the ligation of T7 DNA ligase using ATP or ATPαSe as cofactors at +1 and -1 sites; C. the ligation of T4 DNA ligase using ATP or ATPαSe as cofactors at +1 and -1 sites.)

[0040] Figure 3 In this invention, ATPαSe improves the ability to distinguish single-point mismatches at sites -4 to -1 (A) and +1 to +4 (B).

[0041] (Among them, A. -4 to -1 unit mismatch ligation; B. +4 to +1 unit mismatch ligation; C. the factor by which ATPαSe improves fidelity in ligation reactions relative to natural ATP.)

[0042] Figure 4 This invention utilizes ATPαSe-assisted end ligation for high-throughput sequencing analysis. ATPαSe improved the fidelity of ligation sequences across 93% of the 256 possible ligation types.

[0043] (Among them, A. the ligation sequencing process; B. the change in ligation fidelity for each ligation sequence type compared to ATP-assisted ligation using ATPαSe.)

[0044] Figure 5 This invention relates to the application of ATPαSe in enhancing the fidelity of T4 DNA ligase to increase the positive rate of seamless clones assembled in Kinmen.

[0045] (Where, A. Schematic diagram of the Jinmen assembly used for gene synthesis, through which a plasmid expressing GFP is constructed. BC. Fluorescence analysis of E. coli clones transformed with plasmids assembled with 9 fragments (B) and 15 fragments (C); "Positive rate" indicates the percentage of GFP-positive clones (successfully assembled). (The number of independent replicates for the 9-fragment assembly experiment is 3; the number of independent replicates for the 15-fragment assembly experiment is 10)).

[0046] Figure 6 This invention relates to the application of ATPαSe in enhancing the fidelity of T4 DNA ligase, and its use in increasing the accuracy of MLPA-based multi-SNP detection.

[0047] (Among them, A. Schematic diagram of the ATPαSe-assisted MLPA (Se-MLPA) gene mutation detection scheme; B. In a single MLPA reaction, the use of ATPαSe can significantly reduce false positive signals).

[0048] Figure 7 This invention demonstrates that ATPαSe improves RNA ligation fidelity.

[0049] (Among them, A. Schematic diagram of the ligation substrate and ligation splint sequence; B. ATPαSe improves the accuracy of T4 DNA ligase in ligating RNA fragments). Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments:

[0051] Example 1

[0052] Theoretical analysis of the effect of ATPαSe on ligases:

[0053] Each connection event consists of three consecutive responses. Figure 1 C) In these catalytic steps, mismatches in the nicked substrate DNA can distort the geometry of the active site, affecting the substrate-enzyme affinity and enzyme reaction rate, thus enabling mismatch recognition. Since adenosine-activated intermediates play a crucial role in maintaining the active conformation, including those in mismatch linkages, we believe that modifications to ATP cofactors (including atomic substitution modifications) could potentially regulate interactions with mismatched substrates, thereby modulating mismatch recognition.

[0054] Since selenium and oxygen belong to the same group, the applicant's previous research has confirmed that selenium substitution for oxygen in nucleic acids does not significantly affect the molecular structure and overall molecular function. However, considering the significant differences between selenium and oxygen in the number of electron shells and electron density, the applicant speculates that this atom substitution will greatly affect the metal coordination, hydrogen bonding, and other interactions of that atom. Figure 1 B), thus enabling precise regulation of molecular properties and interactions. Therefore, the applicant hypothesizes that modifying ATP (ATPαSe; ATPαSe contains two diastereomers, and this project uses Peak 1, which can be recognized by ligases;) with selenium atoms. Figure 1 A) As a cofactor for ligases, it may amplify structural distortions and differences in catalytic activity at mismatch sites, thereby inhibiting mis-ligation and enhancing mis-specificity, such as... Figure 1 As shown in C.

[0055] Example 2

[0056] Analysis of the effect of ATPαSe on DNA ligation specificity:

[0057] The standard ligation assay mixture consisted of 0.2 mM ATPαSe, 1× ligase buffer (40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM DTT), T4 DNA ligase (final concentration 50 nM–400 nM), and double-stranded DNA substrate (final concentration 200 nM–1000 nM). Substrate information is shown in Table 1. The reaction was carried out at 37 °C for 30 min, followed by termination with an equal volume of loading buffer (40 mM EDTA, 8 M urea, 30% glycerol, and 0.01% xylenecyanide FF). Separation was finally performed by denaturing polyacrylamide gel electrophoresis (urea-PAGE) containing 8 M urea. The substrate DNA used for ligation was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 1.

[0058] Table 1. DNA substrates used for ligation and their corresponding sequences.

[0059]

[0060] Experiment 1: ATPαSe enhances the fidelity of various DNA ligases

[0061] DNA ligation was performed using ATP (unmodified) and ATPαSe as described in this invention, respectively. The DNA ligation reaction was as described in Example 2, wherein the DNA ligases were T4 DNA ligase, T3 DNA ligase, and T7 DNA ligase, respectively.

[0062] The ligation template used in the ligation reaction is SEQ-1, the ligation substrate acceptor chain is SEQ-2, and the donor chain is SEQ-3 (the 5' end of the donor chain is phosphorylated).

[0063] The beneficial effects of connection include Figure 2 As shown in the figure. The ligation products are displayed on a gel electrophoresis imaging system after passing through a urea-denaturing polyacrylamide gel electrophoresis.

[0064] Figure 2 The following is a verification of the universality of ATPαSe in improving the fidelity of DNA ligases. A shows the ligation results of T3 DNA ligase when mismatches exist between the +1 and -1 sites of the substrate, using ATP or ATPαSe as cofactors; B shows the ligation results of T7 DNA ligase when mismatches exist between the +1 and -1 sites of the substrate, using ATP or ATPαSe as cofactors; and C shows the ligation results of T4 DNA ligase when mismatches exist between the +1 and -1 sites of the substrate, using ATP or ATPαSe as cofactors.

[0065] In this experiment, it was found that the ligation fidelity of T3, T4, and T7 DNA ligases was improved when ATPαSe was used as a cofactor. The degree to which ATPαSe improved the fidelity of different DNA ligases varied, depending on the type of DNA ligase and the location of the mismatch. This invention suggests that ATPαSe can inhibit DNA ligase from ligating substrates containing mismatches, thereby improving the fidelity of DNA ligases.

[0066] Experiment 2: Increased ATPαSe levels allowed ligases to distinguish a wider range of substrate mismatches.

[0067] The DNA ligation reaction is as described in Example 2. The ligation template used for the -4 to -1 mismatch assay is the SEQ-4 sequence. The donor substrate strand is SEQ-5 (5' end modified with phosphate, 3' end modified with FAM fluorescent group). The acceptor substrate strand is one of SEQ-6 (correct match), SEQ-7 (-1 mismatch), SEQ-8 ​​(-2 mismatch), SEQ-9 (-3 mismatch), or SEQ-10 (-4 mismatch). The ligation template used for the +4 to +1 mismatch assay is the SEQ-4 sequence. The donor substrate strand is SEQ-11 (5' end modified with phosphate, 3' end modified with FAM fluorescent group). The acceptor substrate strand is SEQ-12. The ligation template is one of SEQ-13 (correct match), SEQ-14 (+1 mismatch), SEQ-15 (+2 mismatch), SEQ-16 (+3 mismatch), or SEQ-17 (+4 mismatch). The ligation products were displayed on a gel electrophoresis imaging system after passing through urea-denaturing polyacrylamide gel electrophoresis.

[0068] The beneficial effects of the plan are as follows Figure 3 As shown in the image. Figure 3 A enhances the ability to detect mismatches at single sites from -4 to -1 for ATPαSe; Figure 3 B enhances the ability of ATPαSe to distinguish single-site mismatches at sites +1 to +4. Figure 3 C is Figure 3 A and Figure 4 In B, ATPαSe increases fidelity by a factor of 1.

[0069] The results showed that ATPαSe significantly enhanced the mismatch recognition ability in the ligation reaction. In the presence of ATPαSe, almost no mismatch ligation product was formed at site -1, resulting in a 98-fold increase in specificity compared to ATP-assisted ligation. Furthermore, ATPαSe-assisted ligation demonstrated mismatch recognition ability across a wider range, from site -4 to +4, while ATP-assisted ligation failed to effectively recognize mismatches. These results indicate that ATPαSe increases the range of substrate mismatches that ligases can distinguish.

[0070] Experiment 3: ATPαSe enhances specificity in almost all terminal sequence ligations

[0071] Analysis of NGS sequencing and ligated DNA libraries:

[0072] Bottom material library design: such as Figure 4As shown in Figure A, two types of DNA ligation substrates with 4-nt 3'-hangovers (substrate 1 and substrate 2) were designed. Substrate 1 (S1) is a double-stranded structure containing random 4-nt 3'-hangovers, a constant region, and a primer-binding region. Substrate 2 (S2) is a single-stranded hairpin structure containing random 4-nt 3'-hangovers, a constant region, and an additional 13-nt random region (as a barcode to eliminate post-ligation bias). The hanging nucleotides in S1 are named N... -1 To N -4 Used for 3'-overhang connections.

[0073] Substrate library ligation: The ligation reaction was performed at 37°C using 1.0 μM substrate, 1.4 μM T4 DNA ligase, and 1× ligation buffer, in a total volume of 50 μL, for 2 hours. The reaction was terminated by adding 2.5 μL of 500 mM EDTA.

[0074] Analysis of the ligated DNA library: The ligation products were purified by agarose gel electrophoresis and used as templates for PCR amplification. PCR products were purified using a DNA purification kit (Omega, USA). For each sequencing read, barcodes and ligated dangling sequences were extracted. The base composition of each position in the dangling nucleotides was calculated, and the proportion of each nucleotide at each position was determined.

[0075] The beneficial effects of this experiment are as follows: Figure 4 As shown. Figure 4 For high-throughput sequencing analysis of ATPαSe-assisted end ligations, when using ATPαSe and ATP to ligate 3'-droop substrates, ATPαSe demonstrated a significantly higher ability to distinguish mismatch ligations compared to ATP in almost all substrate ligations with 3'-droops. Specifically, among all 245 3'-droop sequence types involving mismatch ligations, 228 ligations showed higher fidelity (93%).

[0076] Example 3

[0077] Application of ATPαSe in enhancing DNA ligase specificity in gene assembly in Kinmen

[0078] Kinmen Assembly Test:

[0079] DNA fragments containing the BsaI recognition site were obtained by PCR (the fully assembled product contained the T7 RNA polymerase gene and the green fluorescent protein gene; sequences are shown in Table 2), and purified using a DNA cleaning kit (Omega, USA). For the Kinmen assembly reaction, each DNA fragment was treated with 100 ng, 0.25 μM T4 DNA ligase, and 15 U BsaI, with a final volume of 20 μL, and 0.2 mM ATP or ATPαSe. The reaction was incubated at 37°C for 2 hours, followed by enzyme inactivation at 55°C for 20 minutes.

[0080] Product transformation into E. coli:

[0081] Add 2 μL of the assembly mixture to 50 μL of competent DH5α cells. Incubate the cells on ice for 30 min, followed by heat shock at 42 °C for 60 s, and then return them to ice for 5 min. Add 950 μL of LB medium and incubate with shaking at 37 °C for 1 h. Spread 200 μL of the sample onto preheated agar plates and incubate at 37 °C for 18 h.

[0082] Analysis of fluorescent expression in clonal bacteria:

[0083] Plates with colonies were imaged and photographed under UV light, and ImageJ was used to count GFP-positive and total colonies. For each assembly type, the total number of transformants and the percentage of correctly assembled (green colonies) were reported as the average of three (9-fragment assembly) or ten (15-fragment assembly) independent replicates.

[0084] Table 2 shows the dsDNA fragments used as substrates for fragment assembly.

[0085]

[0086]

[0087] (Note: In the Kinmen assembly, the overhanging sequences generated by BsaI digestion are underlined.)

[0088] Table 3. dsDNA fragments used as substrates for fragment assembly (Table 35)

[0089]

[0090]

[0091] (Note: In the Kinmen assembly, the overhanging sequences generated by BsaI digestion are underlined.)

[0092] The beneficial effects of this experiment are as follows: Figure 5 As shown in the image. Figure 5This study describes the application of ATPαSe to improve the accuracy of T4 DNA ligase in the assembly of seamless clones in Jinmen. Figure A shows a schematic diagram of Jinmen assembly for gene synthesis. Accurately assembled plasmids can express T7 RNA polymerase after transfection into cells. T7 RNA polymerase then initiates green fluorescent protein (GFP) expression, ultimately causing the cloned bacteria to exhibit green fluorescence under excitation light. Figures B and C show the fluorescence analysis of *E. coli* clones transformed with plasmids assembled from 9 fragments (B) and 15 fragments (C). "Positive rate" indicates the percentage of GFP-positive clones. (The 9-fragment assembly included 3 independent replicates; the 15-fragment assembly included 10 independent replicates).

[0093] Experimental results showed that in the synthesis of 9-fragment genes, ATPαSe provided a higher proportion of positive clones (green fluorescence positive; 41.3%) compared to ATP (7.7%). Furthermore, in the synthesis of 15-fragment genes, ATPαSe provided a significantly higher proportion of positive clones (22.2%; more than 200-fold increase) compared to ATP (0.1%). Therefore, in gene synthesis involving a large number of fragments and where random selection of overhang sequences (even including error-prone sequences) is challenging, conventional ATP is almost unable to produce positive clones. In contrast, ATPαSe not only promotes assembly and allows for a wider selection of overhang sequences but also significantly increases the proportion of positive clones.

[0094] Example 4

[0095] ATPαSe enhances the application of DNA ligase in the detection of SNPs using multiplex ligation-dependent probe amplification.

[0096] ATPαSe-assisted multiplex ligation-dependent probe amplification (Se-MLPA) for SNP detection:

[0097] To evaluate the discriminative ability of Se-MLPA in SNP detection, an MLPA reaction was designed using a pair of probes (3'-probe and 5'-probe) to detect gene SNPs. The 3'-probe contains a primer recognition region and a hybridization region (for target pairing); the 5'-probe contains an additional filler sequence for product differentiation during electrophoresis. Six pairs of probes were used specifically for six SNPs in six mutant genes. All probes and templates were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 1 (SEQ-42–SEQ-65). The reaction was performed by mixing the probes (0.4 μM each) and template mixtures (0.4 μM each, containing either six mutant or six wild-type templates) in 1× ligation buffer (40 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM DTT). A denaturation-annealing process was performed by slowly cooling from 95 °C to room temperature to promote hybridization. The ligation reaction was initiated by adding 5 nM T4 DNA ligase and 5 μM ATP (or ATPαSe), followed by incubation at 37 °C for 5 minutes. Subsequently, the ligation product was diluted and PCR amplified for 20 cycles using universal primers PriX (SEQ-66) and PriY (SEQ-67) (95 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds). The PCR products were analyzed by 4% agarose gel electrophoresis.

[0098] The beneficial effects of this experiment are as follows: Figure 6 As shown. Figure 6 This paper describes the application of ATPαSe to improve the accuracy of T4 DNA ligase in the detection of multiple single-base mutation nucleic acids. A shows a schematic diagram of the ATPαSe-assisted MLPA (Se-MLPA) gene mutation detection scheme, where multiple mutation detection achieves separation through spacer segments of different lengths. B illustrates the simultaneous detection of six SNP targets using mutation-targeting probes and ATP or ATPαSe in the MLPA reaction.

[0099] This invention explores the ATPαSe substitution strategy using single nucleotide polymorphism (SNP) detection as an example and designs a six-fold MLPA reaction (…). Figure 6 A), used to detect six SNPs in six mutant genes. This invention found that ATPαSe-assisted MLPA (Se-MLPA) exhibits good discriminative ability in suppressing background noise caused by the wild-type gene template (WT-Tem). Figure 6 B). Se-MLPA produced a true positive signal (six bands), while MLPA using ATP failed to suppress background false positive signals. The results indicate that the ATPαSe substitution strategy of this invention can significantly improve the accuracy of ligase-based detection methods by distinguishing between true and false signals and suppressing background noise.

[0100] Table 4. Probe and primer sequences used for multiplex ligation-dependent probe amplification.

[0101]

[0102]

[0103] Example 5

[0104] Analysis of the effect of ATPαSe on RNA linker specificity:

[0105] T4 DNA ligase can ligate two RNA fragments in a DNA clip-assisted ligation setting and can also be considered an RNA ligase. This experiment analyzed the effect of ATPαSe on the RNA ligation specificity of T4 DNA ligase. The RNA ligation assay mixture consisted of 0.1 mM ATPαSe, 1× ligase buffer (40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM DTT), T4 DNA ligase (0.7 μM for ATP-assisted ligation; 2.8 μM for ATPαSe-assisted ligation), and RNA substrate (0.1 μM for ATP-assisted ligation; 0.005 μM for ATPαSe-assisted ligation). DNA clip information is shown in Table 5, and RNA substrate sequences are shown in Table 6. The reaction was carried out at 37°C for 2 h (ATP-assisted ligation assay) or 5 h (ATPαSe-assisted ligation assay), and then terminated with an equal volume of loading buffer (40 mM EDTA, 8 M urea, 30% glycerol and 0.01% xylenecyanine FF). Finally, the reaction was analyzed and purified by denaturing polyacrylamide gel electrophoresis (urea-PAGE) containing 8 M urea.

[0106] Table 5 DNA clips for RNA ligation

[0107]

[0108] Table 6. RNA substrates for RNA ligation

[0109]

[0110] The success of our solution is as follows: Figure 7 As shown in the image. Figure 7 A is a schematic diagram of the sequence of connecting the substrate and the connecting clamps; Figure 7 B shows the PAGE electrophoresis results demonstrating how ATPαSe enhances the accuracy of T4 DNA ligase in ligating RNA fragments.

[0111] The results showed that ATPαSe significantly enhanced the ligase's ability to correctly recognize base mismatches in RNA ligation: the recognition specificity of the reaction was significantly improved (1.1-7.0 times) for base mismatches from +1 to +4. Especially for the ligation of base mismatches at +2, almost no ligation product was formed in the presence of ATPαSe, and the ligation specificity of ATPαSe-assisted ligation was 7.0 times higher than that of ATP-assisted ligation.

[0112] The above embodiments / experimental examples are merely illustrative and not intended to limit the implementation methods. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementation methods. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. The use of selenium-modified ATPαSe as a DNA-linking cofactor and an RNA-linking cofactor, characterized in that: The application involves using ATPαSe to replace natural ATP as a cofactor for ligases in DNA and RNA ligation and DNA-RNA hybrid ligation based on nucleic acid ligation templates. This enhances the ability of DNA ligases and RNA ligases to distinguish base mismatches between the ligation template and the ligation substrate, and helps improve their ability to recognize correct base matches. This improves the accuracy of DNA ligases and RNA ligases, and can also be used to improve the accuracy of single-base mutation detection based on DNA ligases, the accuracy of seamless cloning in Kinmen assembly, and the accuracy of multiplex ligation-dependent probe amplification technology.

2. The use of the selenium-modified ATPαSe cofactor according to claim 1, characterized in that: The DNA ligase is a T4 DNA ligase, a T3 DNA ligase, a T7 DNA ligase, or another DNA ligase that uses ATP as a cofactor.

3. The use of the selenium-modified ATPαSe cofactor according to claim 1, characterized in that: The RNA ligase is T4 DNA ligase or other RNA ligases that use ATP as a cofactor.

4. The use of a selenium-modified ATPαSe cofactor according to claims 1, 2, and 3, characterized in that: The dosage of the ATPαSe cofactor is 1 μM to 50 mM.

5. The use of a selenium-modified ATPαSe cofactor according to claims 1, 2, and 3, characterized in that: The dosage of the ATPαSe cofactor is 0.2 mM.

6. The use of the ATPαSe cofactor according to claims 1 and 2 in the preparation of reagents for the detection of ligation-dependent multiplex ligation-dependent probe amplification.

7. The use of the ATPαSe cofactor as described in claims 1 and 2 in the preparation of reagents for assembling seamless clones in Kinmen.

8. A method for improving the fidelity of DNA and RNA ligation and DNA-RNA hybrid ligation based on selenium-modified ATP, characterized in that: This includes applying ATPαSe cofactors, as in claims 1 and 2, to the DNA ligation reaction system to enhance the mismatch recognition ability of DNA ligase; or its application in the assembly of seamless clones in Kinmen; or its application in the detection of multiple ligation-dependent probe amplification.

9. The method according to claim 8 is characterized in that: The DNA or RNA or DNA-RNA mixed ligation reaction system is a mixture including ATPαSe ligation cofactor.

10. The method according to claim 8, characterized in that: The reaction time is 1 min to 48 h and the temperature is 4 °C to 45 °C.

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