Method for improving cyclization yield of threose nucleic acid
By adding a G base to the 5' end of DNA and an A base to the 2' end of TNA, the cyclization reaction conditions of threonine nucleic acid were optimized, solving the problem of low cyclization yield, improving cyclization efficiency, and expanding the application potential of threonine nucleic acid in the field of biotechnology.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING UNIV
- Filing Date
- 2026-02-12
- Publication Date
- 2026-05-08
AI Technical Summary
In existing technologies, the cyclization yield of threonine (TNA) is low, which limits its application potential in the field of biotechnology.
By optimizing cyclization reaction conditions, including temperature and time, through the strategy of adding a G base to the 5' end of DNA and an A base to the 2' end of TNA, the cyclization yield was improved.
Significant improvements were achieved in the 12-hour cyclization yield of 16 sequences, with some sequences exceeding 70%, and the 2-hour cyclization yield increased by up to 30-fold, thus improving the cyclization efficiency of threonine.
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Figure CN121992053A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of nucleic acid technology and non-natural nucleic acid applications, and relates to a method for improving the cyclization yield of threonine nucleic acids; specifically, it relates to a method that achieves a cyclization yield of more than 70% after adding one G base to the 5' end of the DNA sequence and one or two A bases to the 2' end of the TNA sequence. Background Technology
[0002] In recent years, circular nucleic acids (CNs) have attracted widespread attention in biological research due to their unique circular structure. This structure eliminates the 5' and 3' ends of linear nucleic acids, significantly enhancing their resistance to degradation by nucleases and resulting in a longer half-life and higher stability within cells. Furthermore, CN aptamers often exhibit good structural rigidity and highly specific target binding capabilities, demonstrating significant potential in drug delivery, targeted therapy, and biosensing. Meanwhile, circularized DNA templates can also be used for rolling circle amplification, further applied to disease diagnosis and biosensor development. Threonine nucleic acid (TNA) is an artificial nucleic acid composed of α-L-threonanose molecules linked by 2',3'-phosphodiester bonds, with a simpler backbone structure than DNA and RNA. TNA not only possesses excellent biological stability but can also form stable double-stranded structures with DNA and RNA, enabling the exchange of genetic information, thus attracting considerable attention in the study of the origin of life and the development of molecular tools. With in-depth exploration of its functions, various TNA molecules have been developed, including those targeting trachomacin A, binding to HIV reverse transcriptase, and possessing RNA endonuclease activity, showing broad application prospects.
[0003] In the existing technology, there is a method for cyclizing TNA using CircLigase, but the main drawback of this method is the low yield of partial sequence cyclization, which limits its potential for expanding the field of biotechnology. Summary of the Invention
[0004] Purpose of the invention: The purpose of this invention is to provide a method for improving the threonine cyclization yield by adding a G base to the 5' end of the DNA sequence and an A base to the 2' end of the TNA sequence.
[0005] The technical solution of the present invention is: a method for improving the cyclization yield of threonine nucleic acids, comprising the following steps: Step (1) Using a DNA 5' end G base and a TNA 2' end A base sequence as substrate, the cyclization yield was observed by changing the time and temperature of the substrate cyclization reaction. The 12-h cyclization yield of 16 different end combination sequences was also investigated. These 16 sequences were named the initial sequences, which are 4×4 combinations of DNA 5' end A, T, G, C bases and TNA 2' end A, T, G, C bases. Among them, only two initial sequences had a circularization yield of ≥70% after 12 h. These two combinations were a DNA 5' end G base and a TNA 2' end A base sequence, and a DNA 5' end G base and a TNA 2' end G base sequence. Step (2) For 14 different terminal base combinations with low cyclization yield, add 1 G base to the 5' end of the DNA and 1 A base to the 2' end of the TNA, and study the effect of this strategy on cyclization yield; Among them, the cyclization yield of 8 sequences increased to ≥70% after 12 h, while the cyclization yield of the other 6 sequences was between 40% and 60%. Step (3) Based on step (2), after adding one more A base to the 2' end of TNA, the other 6 sequences with cyclization yields of 40-60% were optimized again, so that the cyclization yields of the optimized sequences were all increased to ≥70% after 12 h; Step (4) compare the 2-hour cyclization yield of the 14 initial sequences with low cyclization yields with their corresponding final optimized sequences. Compared with the initial sequences, the 2-hour cyclization yields of the optimized sequences are significantly improved, with the highest improvement being 30 times. The final optimized sequences are the 8 sequences in step (2) whose cyclization yield was increased to ≥70% at 12 h, and the 6 sequences optimized in step (3). The 2 h time point was chosen because the reaction is in progress at this time, which can more accurately show the improvement effect of the optimization strategy.
[0006] Further, step (1) specifically involves: First, adding CircLigase, corresponding buffer, ATP, and MnCl2, using a DNA-TNA chimeric single strand modified with monophosphate and Cy5.5 fluorophores at the 5' end and inside the DNA, respectively, and with a G base at the 5' end of the DNA and an A base at the 2' end of the TNA as the substrate, reacting at 55°C for different times, and reacting at different temperatures for 2 h, measuring the fluorescence intensity of the remaining single strand by denaturing polyacrylamide gel electrophoresis and fluorescence imaging system, and calculating the cyclization yield; reacting 16 different end combination sequences at 55°C for 12 h, with other conditions unchanged, and calculating the cyclization yield; The cyclization reaction of the DNA sequence with a G base at the 5' end and the TNA sequence with an A base at the 2' end showed the highest cyclization yield at 55°C, reaching 84% after 3 hours. Among the 16 initial sequences, only 2 had a cyclization yield ≥70% after 12 hours.
[0007] Furthermore, step (2) specifically involves adding one G base to the 5' end of the DNA and one A base to the 2' end of the TNA for 14 different terminal base combinations with a cyclization yield of less than 70% over 12 hours. If the original 5' end is a G base or the 2' end is an A base, the corresponding ends remain unchanged. The optimized sequences are then cyclized at 55°C for 12 hours using CircLigase catalysis. The cyclization yield of 8 sequences is increased to ≥70%, while the cyclization yield of the other 6 sequences is between 40-60%.
[0008] Furthermore, in step (3), for the six sequences with a cyclization yield of 40-60% after optimization in step (2), one A base is added to the 2' end of the TNA, that is, one G base is added to the 5' end of the initial DNA sequence and two A bases are added to the 2' end of the TNA. The cyclization yield of the optimized sequences is improved, and the cyclization yield increases to ≥70% after 12 h of reaction.
[0009] Furthermore, specifically: First, based on the six sequences with a cyclization yield of only 40-60% in step (2), add one A base to the 2' end of TNA; order the corresponding template and primer sequences, with primers containing Cy5.5 fluorophores and modified with monophosphate; Next, the primers were extended with TNA on the corresponding template to prepare DNA-TNA chimeric single strands, which were then purified. Then, for each type of DNA-TNA chimeric single strand, the same concentration and volume were added to CircLigase, the corresponding buffer, ATP, and MnCl2, so that CircLigase catalyzed the reaction of the DNA-TNA chimeric single strand in the buffer. The reaction was stopped after reacting at 55°C for 12 h. Finally, the fluorescence intensity of the remaining single strands was measured by denaturing polyacrylamide gel electrophoresis and fluorescence imaging system. It was found that after adding one A base to the 2' end of the TNA of the six sequences, the cyclization yield increased to ≥70% after 12 h of reaction.
[0010] Further, step (4) specifically involves: selecting the initial sequence and the corresponding final optimized sequence, taking the same concentration and volume of CircLigase, the corresponding buffer, ATP, and MnCl2, so that CircLigase catalyzes the reaction of DNA-TNA chimeric single strands in the buffer, and stopping the reaction after reacting at 55°C for 2 h. The amount of remaining single strands was determined by denaturing polyacrylamide gel electrophoresis, and the cyclization yield of each sequence was calculated. The changes in cyclization yield before and after optimization were compared by plotting bar graphs. It was found that after optimization by adding bases to the final end, the cyclization yield of all optimized final sequences was significantly improved after 2 h compared with the initial sequence, with the maximum improvement being 30-fold.
[0011] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention determines the optimal temperature and time for CircLigase cyclization of DNA sequences with a G base at the 5' end and an A base at the 2' end of TNA. By using a strategy of adding one G base at the 5' end of DNA and one A base at the 2' end of TNA, the invention optimizes 14 sequences with low cyclization yields, increasing the cyclization yield of 8 sequences to 80% within 12 hours and 6 sequences to 40-60%. Further addition of one A base at the 2' end of TNA further increases the cyclization yield of these 6 sequences to approximately 80% within 12 hours. This invention provides a promising strategy for the application of nucleic acid technology and non-natural nucleic acids, especially for enzymatically cyclizing TNA to expand its applications. Attached Figure Description
[0012] Figure 1 This is a flowchart of the operation of the present invention; Figure 2 This is a schematic diagram showing the change in the circularization yield of DNA-TNA chimeric single strands with a G base at the 5' end and an A base at the 2' end in different times or temperatures in Example 1 of the present invention. Figure 3 This is a comparison of the 12-hour circularization yield of the sequences after adding one G base to the 5' end of DNA and adding one A base to the 2' end of TNA in Example 1 of the present invention. In the figure, gray represents the unchanged sequence and red represents the optimized sequence. Figure 4 This is a comparison chart of 12-hour cyclization yields of sequences after the further terminal addition strategy in Example 2 of the present invention. In the chart, the blue part represents the sequence in which one A base is added to the 2' end of TNA based on the first step. Figure 5 This is a comparison graph of the 2h cyclization yields of the initial sequence and the final optimized sequence in Embodiment 2 of the present invention. Detailed Implementation
[0013] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.
[0014] like Figure 1 As shown, the method for improving the cyclization yield of threonine nucleic acid according to the present invention includes the following steps: Step (1) evaluated the effects of time and temperature on the cyclization yield of DNA sequences with G bases at the 5' end and A bases at the 2' end. It was found that the cyclization yield was highest at 55°C, and the cyclization yield could reach 84% after 3 h. Step (2) Add one G base to the 5' end of the DNA of the sequence with low circularization yield and add one A base to the 2' end of the TNA. The circularization yield of the optimized sequence was significantly improved after 12 h, but some sequences only had about 40-60%. In step (3), after adding one G base to the 5' end of the DNA and one A base to the 2' end of the TNA, the final optimized sequence achieved a cyclization yield of over 70% after 12 hours. Step (4) compare the 2-h circularization yield of the 16 initial sequences and the final optimized sequences. The circularization yield of all optimized sequences was significantly improved compared with the initial sequences, with the maximum improvement being 30 times.
[0015] In step (1), CircLigase was used to catalyze the cyclization reaction of DNA-TNA chimeric single strands with G base at the 5' end of DNA and A base at the 2' end of TNA at different times and temperatures. The optimal reaction time and temperature were evaluated. The cyclization yield was highest at 55℃, and the cyclization yield could reach 84% after 3 h of reaction.
[0016] In step (2), for 14 different terminal base combinations with a cyclization yield of less than 70% after 12 h, one G base was added to the 5' end of the DNA and one A base was added to the 2' end of the TNA. If the original 5' end was a G base or the 2' end was an A base, the corresponding ends remained unchanged. The optimized sequences were cyclized at 55 °C for 12 h using CircLigase catalysis. The cyclization yield of 8 sequences was increased to more than 70%, and the cyclization yield of the other 6 sequences was increased to 40-60%.
[0017] Based on step (3), for the six sequences with low cyclization yields after optimization in (2), one A base was added to the 2' end of the TNA, that is, one G base was added to the 5' end of the initial DNA sequence and two A bases were added to the 2' end of the TNA. The cyclization yields of the optimized sequences were significantly improved, and more than 70% could be cyclized after 12 h of reaction.
[0018] In step (4), the 16 initial sequences and their corresponding final optimized sequences were cyclized by CircLigase catalysis and reacted at 55°C for 2 h. Compared with the initial sequences, the final optimized cyclization yield was increased by about 30 times.
[0019] Example 1: As shown in the figure, the change in cyclization yield of a DNA-TNA chimeric single strand with a G base at the 5' end and an A base at the 2' end of the TNA over time and at different temperatures was identified. 1. Sequences used in synthesis: Single-stranded DNA primers with double modification of monophosphate and fluorophore: 5'-pGTTTG / iCY5.5dT / GAGTAATTGTTATT-3'; Single-stranded DNA template: 5'-TACTTAACTATACCAATCCTAATCTCAGAGACTCTACTCAATAACAATTACTCACAAAC-3'; 2. The TNA polymerase Kod-RSGA, purified and expressed in the laboratory, and four TNA triphosphate monomers were used to extend the DNA at the 3' end of single-stranded DNA. The reaction system contained 0.1 μM single-stranded DNA primers, 0.1 μM single-stranded DNA template, 0.1 mM TNA triphosphate monomers, 1× ThermoPol reaction buffer (100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 200 mM Tris-hydrochloric acid, 0.1% Triton X-100, pH 8.8), and 1 mg / mL Kod-RSGA. After extension at 55℃ for 4 h, the DNA was precipitated with ethanol and purified by gel electrophoresis to obtain the DNA-TNA chimeric single strands. 3. Divide the obtained DNA-TNA chimeric single strands into several equal portions, each containing 1 pmol of DNA-TNA chimeric single strand. In the reaction buffer, use CircLigase to catalyze the cyclization reaction at 55°C for 0, 5, 10, 30, 60, 180, and 480 min, and at 4, 25, 37, 45, 55, 65, and 75°C for 2 h, respectively. The reaction system contains 0.05 μM DNA-TNA chimeric single strands, 2.5 mM MnCl2, 0.05 mM ATP, 1× CircLigase reaction buffer, and 0.05 mg / mL CircLigase. The control group does not contain CircLigase, and the rest is the same as the experimental group. Then add 2 volumes of stop buffer (7M urea dissolved in 1× TBE). 4. Each group of samples was characterized by 12% denaturing polyacrylamide gel electrophoresis. After electrophoresis at a constant power of 100 W for 1 h, the fluorescence intensity of the bands was measured using an Odyssey CLx dual-color infrared laser imaging system (700 nm excitation channel). The cyclization yield was quantified by the ratio of the fluorescence intensity of the cyclized single-chain bands to that of the cyclized plus linear single-chain bands. 5. Plot a graph showing the change in the cyclization yield of DNA-TNA chimeric single strands over time to evaluate the cyclization yield of DNA-TNA chimeric single strands catalyzed by CircLigase with a G base at the 5' end of DNA and an A base at the 2' end of TNA. 6. Finally, it was determined that the cyclization yield increased continuously with time, reaching 84% at 3 h; the cyclization yield first increased and then decreased with temperature, with the highest cyclization yield at 55℃ at 2 h.
[0020] Example 2: 12-hour cyclization yield of DNA with 1 G base added to the 5' end and TNA with 1 A base added to the 2' end: 1. Sequences used in synthesis Single-stranded DNA primers with dual modification of monophosphate and fluorophore: 5'-pGTTTG / iCY5.5dT / GAGTAATTGTTATT-3'; 5'-pGATTTG / iCY5.5dT / GAGTAATTGTTATT-3'; 5'-pGTTTTG / iCY5.5dT / GAGTAATTGTTATT-3'; 5'-pGCTTTG / iCY5.5dT / GAGTAATTGTTATT-3'; Single-stranded DNA template: 5'-TACTTAACTATACCAATCCTAATCTCAGAGACTCTACTCAATAACAATTACTCACAAAC-3'; 5'-TAACTTAACTATACCAATCCTAATCTCAGAGACTCTACTCAATAACAATTACTCACAAAC-3'; 5'-TGACTTAACTATACCAATCCTAATCTCAGAGACTCTACTCAATAACAATTACTCACAAAC-3'; 5'-TCACTTAACTATACCAATCCTAATCTCAGAGACTCTACTCAATAACAATTACTCACAAAC-3'; 2. The TNA polymerase Kod-RSGA, purified and expressed in the laboratory, and four TNA triphosphate monomers were used to extend the DNA at the 3' end of single-stranded DNA. The reaction system contained 0.1 μM single-stranded DNA primers, 0.1 μM single-stranded DNA template, 0.1 mM TNA triphosphate monomers, 1× ThermoPol reaction buffer (100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 200 mM Tris-hydrochloric acid, 0.1% Triton X-100, pH 8.8), and 1 mg / mL Kod-RSGA. After extension at 55℃ for 4 h, the DNA was precipitated with ethanol and purified by gel extraction to obtain DNA-TNA chimeric single strands. 3. Divide the obtained DNA-TNA chimeric single strands into several equal portions, each containing 1 pmol of DNA-TNA chimeric single strand. In the reaction buffer, perform a cyclization reaction catalyzed by CircLigase at 55°C for 12 h. The reaction system contains 0.05 μM DNA-TNA chimeric single strands, 2.5 mM MnCl2, 0.05 mM ATP, 1× CircLigase reaction buffer, and 0.05 mg / mL CircLigase. Then add 2 volumes of stop buffer (7 M urea dissolved in 1× TBE). 4. Each group of samples was characterized by 12% denaturing polyacrylamide gel electrophoresis. After electrophoresis at a constant power of 100 W for 1 h, the fluorescence intensity of the bands was measured using an Odyssey CLx dual-color infrared laser imaging system (700 nm excitation channel). The cyclization yield was quantified by the ratio of the fluorescence intensity of the cyclized single-chain bands to that of the cyclized plus linear single-chain bands. 5. Plot bar charts of different DNA-TNA chimeric single-strand circularization yields to evaluate the impact of end-addition strategies on DNA-TNA chimeric single-strand circularization yields; 6. Finally, it was found that the 12-hour cyclization yield of the 8 optimized sequences reached about 80%, and the cyclization yield of the 6 sequences was between 40-60%.
Claims
1. A method for improving the cyclization yield of threonine nucleic acids, characterized in that, Includes the following steps: Step (1) Using a DNA 5' end G base and a TNA 2' end A base sequence as a substrate, the cyclization yield was observed by changing the time and temperature of the substrate cyclization reaction. The 12h cyclization yield of 16 different end combination sequences formed by the substrate was investigated. Among them, the 12h cyclization yield of 2 sequences was ≥70%. Step (2) For the other 14 different terminal base combinations with low cyclization yield, add one G base to the 5' end of their DNA and one A base to the 2' end of their TNA, and study the effect of this strategy on cyclization yield; Among them, the cyclization yield of 8 sequences increased to ≥70% after 12 h, while the cyclization yield of the other 6 sequences was between 40% and 60%. Step (3) Based on step (2), after adding one more A base to the 2' end of TNA, the other 6 sequences with cyclization yields of 40-60% were optimized again, so that the cyclization yields of the optimized sequences were all increased to ≥70% at 12 h; Step (4) compare the 2-h cyclization yield of the 14 initial sequences with low cyclization yields with their corresponding final optimized sequences; compared with the initial sequences, the 2-h cyclization yields of the optimized sequences have all been improved to varying degrees.
2. The method for improving the cyclization yield of threonine nucleic acid according to claim 1, characterized in that, The 16 different end combination sequences mentioned in step (1) are initial sequences formed by a 4×4 combination of the A, T, G, and C bases at the 5' end of DNA and the A, T, G, and C bases at the 2' end of TNA.
3. The method for improving the cyclization yield of threonine nucleic acid according to claim 1, characterized in that, The combination of the two sequences in step (1) is a DNA 5' end G base and a TNA 2' end A base sequence, and a DNA 5' end G base and a TNA 2' end G base sequence.
4. The method for improving the cyclization yield of threonine nucleic acid according to claim 1, characterized in that, The specific operation process of step (1) is as follows: CircLigase, corresponding buffer, ATP, and MnCl2 are added to modify the 5' end and the inside of the DNA with monophosphate and Cy5.5 fluorophores, respectively, and the DNA-TNA chimeric single strand with G base at the 5' end and A base at the 2' end of the TNA is used as the substrate. The reaction is carried out at 55°C for different times and at different temperatures for 2 h. The fluorescence intensity of the remaining single strand is measured by denaturing polyacrylamide gel electrophoresis and fluorescence imaging system, and the cyclization yield is calculated.
5. The method for improving the cyclization yield of threonine nucleic acid according to claim 4, characterized in that, The cyclization reaction of sequences with a G base at the 5' end of DNA and an A base at the 2' end of TNA, and a G base at the 5' end of DNA and a G base at the 2' end of TNA, showed the highest cyclization yield at 55°C, reaching 84% after 3 hours.
6. The method for improving the cyclization yield of threonine nucleic acid according to claim 1, characterized in that, The specific steps (2) are as follows: For 14 different terminal base combination sequences with a cyclization yield of less than 70% after 12 h, add 1 G base to the 5' end of the DNA and 1 A base to the 2' end of the TNA. If the original 5' end is a G base or the 2' end is an A base, the corresponding ends remain unchanged. After the optimized sequences are catalyzed by CircLigase, they are cyclized at 55°C for 12 h. The cyclization yield of 8 sequences is increased to ≥70%, and the cyclization yield of the other 6 sequences is between 40-60%.
7. The method for improving the cyclization yield of threonine nucleic acid according to claim 1, characterized in that, In step (3), for the six sequences with cyclization yields of 40-60% after optimization in step (2), one A base is added to the 2' end of the TNA, that is, one G base is added to the 5' end of the initial DNA sequence and two A bases are added to the 2' end of the TNA. The cyclization yields of the optimized sequences are all improved, and the cyclization yield increases to ≥70% after 12 h of reaction.
8. The method for improving the cyclization yield of threonine nucleic acid according to claim 7, characterized in that, Specifically: First, based on the six sequences with a cyclization yield of only 40-60% in step (2), add one A base to the 2' end of TNA; order the corresponding template and primer sequences, with primers containing Cy5.5 fluorophores and monophosphate modifications; Next, the primers were extended with TNA on the corresponding template to prepare DNA-TNA chimeric single strands, which were then purified. Then, for each type of DNA-TNA chimeric single strand, the same concentration and volume were added to CircLigase, the corresponding buffer, ATP, and MnCl2, so that CircLigase catalyzed the reaction of the DNA-TNA chimeric single strand in the buffer. The reaction was stopped after reacting at 55°C for 12 h. Finally, the fluorescence intensity of the remaining single strands was measured by denaturing polyacrylamide gel electrophoresis and fluorescence imaging system. It was found that after adding one A base to the 2' end of the TNA of the six sequences, the cyclization yield increased to ≥70% after 12 h of reaction.
9. The method for improving the cyclization yield of threonine nucleic acid according to claim 1, characterized in that, The specific steps (4) are as follows: Select the initial sequence and the corresponding final optimized sequence, take the same concentration and volume and add CircLigase, the corresponding buffer, ATP and MnCl2, so that CircLigase catalyzes the reaction of DNA-TNA chimeric single strands in the buffer, and stop the reaction after reacting at 55℃ for 2 h. The amount of remaining single strands was determined by denaturing polyacrylamide gel electrophoresis, the cyclization yield of each sequence was calculated, and the changes in cyclization yield before and after optimization were compared by plotting bar graphs. It was found that after optimization by the final end-base addition strategy, the cyclization yield of all optimized final sequences was improved by 2 h compared with the initial sequence.
10. The method for improving the cyclization yield of threonine nucleic acid according to claim 9, characterized in that, The final optimized sequences are the 8 sequences in step (2) with a 12-hour cyclization yield of ≥70% and the 6 sequences optimized in step (3).