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CN-121992053-A - Method for improving cyclizing yield of threose nucleic acid

CN121992053ACN 121992053 ACN121992053 ACN 121992053ACN-121992053-A

Abstract

The invention discloses a method for improving the cyclizing yield of threose nucleic acid, which comprises the specific steps of evaluating the cyclizing yield of a sequence with G base at the 5' end and A base at the TNA 2' end of DNA at different temperatures and times, wherein the cyclizing yield is highest at 55 ℃, the cyclizing yield reaches 84% at 3h, aiming at the problem that the cyclizing yield of a part of terminal base combination sequence 12 h is low, 1G base is added at the 5' end of DNA, 1A base is added at the TNA 2' end of DNA, the cyclizing yield is improved, the cyclizing yield of 12 h of a tested sequence reaches more than 70% after 1A base is further added at the 2' end of DNA, compared with an initial sequence, the cyclizing yield of 2 h of a final optimized sequence is improved by 30 times at most, the influence of the temperature, the time and the types of the two ends of the sequence on the cyclizing yield of TNA is explored, the method for improving the cyclizing yield of TNA through terminal base addition is established and optimized, the application range of CircLigase in TNA cyclizing is widened, and the application range of TNA molecule is facilitated in the field of promoting the cyclizing and the application of TNA molecule in the field of biology.

Inventors

  • Yu hanyang
  • ZOU YE
  • WANG JUAN

Assignees

  • 南京大学

Dates

Publication Date
20260508
Application Date
20260212

Claims (10)

  1. 1. A method for increasing the yield of circularization of a threose nucleic acid, comprising the steps of: Step (1) taking a DNA 5 'end as a G base and a TNA 2' end as a base sequence as a substrate, observing the change of cyclization yield by changing the time and temperature of cyclization reaction of the substrate, and exploring the 12h cyclization yields of 16 different terminal combined sequences synthesized by combining the substrate, wherein the 12h cyclization yield of 2 sequences is more than or equal to 70%; Step (2) for 14 different terminal base combination sequences with low cyclization yields, 1G base is added at the 5 'end of DNA and 1A base is added at the 2' end of TNA, and the influence of this strategy on cyclization yields is studied; Wherein, the cyclization yield of 8 sequences 12 h is increased to be more than or equal to 70 percent, and the cyclization yield of 6 sequences is 40 to 60 percent; step (3) after adding 1A base at the TNA 2' end based on the step (2), optimizing the other 6 sequences with the cyclization yield of 40-60% again, so that the cyclization yield of 12h of the optimized sequences is increased to be more than or equal to 70%; Step (4) compares the cyclization yields of the 14 initial sequences with lower cyclization yields with the cyclization yields of 2h of the corresponding final optimized sequences, and the cyclization yields of the optimized sequences 2h are improved to different degrees compared with the initial sequences.
  2. 2. The method of claim 1, wherein the 16 different terminal combined sequences in step (1) are the initial sequences formed by 4 x 4 combinations of the A, T, G, C bases at the 5 'end of DNA and A, T, G, C bases at the 2' end of TNA.
  3. 3. The method for improving the circularization yield of threose nucleic acid according to claim 1, wherein the combination of the 2 sequences in the step (1) is a sequence of a DNA having a G base at a 5 'end and a TNA 2' end, and a sequence of a DNA having a G base at a 5 'end and a G base at a TNA 2' end.
  4. 4. The method for improving the cyclization yield of threose nucleic acid according to claim 1, wherein the specific operation process of the step (1) is that adding CircLigase, corresponding buffer solution, ATP and MnCl 2 to respectively modify monophosphate and Cy5.5 fluorophores in the 5' end and the DNA, and reacting the DNA-TNA chimeric single strand with G base at the 5' end and A base at the TNA 2' end as substrates for 2h at different temperatures for different time at 55 ℃, and measuring the fluorescence intensity of the residual single strand by a denaturing polyacrylamide gel electrophoresis and fluorescence imaging system to calculate the cyclization yield.
  5. 5. The method for improving the cyclization yield of threose nucleic acid according to claim 4, wherein the cyclization reaction of the sequence with the G base at the 5 'end of DNA and the A base at the 2' end of TNA and the G base at the 5 'end of DNA and the G base at the 2' end of TNA is identified, and the cyclization yield is the highest at 55 ℃, and the cyclization yield of 3 h is 84%.
  6. 6. The method for improving the cyclization yield of threose nucleic acid according to claim 1, wherein the step (2) is characterized in that for 14 different terminal base combination sequences with the cyclization yield of 12 h lower than 70%, 1G base is added to the 5 'end of DNA and 1A base is added to the 2' end of TNA, if the original 5 'end is G base or the 2' end is A, the corresponding ends are unchanged, the cyclization reaction is carried out for the 8 sequences at 55 ℃ by using the CircLigase catalytic optimization, wherein the cyclization yield of the 8 sequences is improved to be more than or equal to 70%, and the cyclization yield of the other 6 sequences is 40-60%.
  7. 7. The method for improving the cyclization yield of threose nucleic acid according to claim 1, wherein in the step (3), for the sequence with the optimized 6 cyclization yields of 40-60% in the step (2), 1 more A base is added at the 2' -end of TNA, namely 1G base is added at the 5' -end of the initial sequence DNA and 2A bases are added at the 2' -end of TNA, the cyclization yields of the optimized sequences are improved, and the cyclization yield is improved to be more than or equal to 70% after the reaction of 12 h.
  8. 8. The method for improving the cyclization yield of threose nucleic acid according to claim 7, wherein the method is characterized in that firstly, 1A base is added at the 2' end of TNA on the basis of 6 sequences with the cyclization yield of only 40-60% in the step (2); Then, the primer is subjected to TNA extension on the corresponding template to prepare a DNA-TNA chimeric single strand, and purification is performed; then, adding the CircLigase, corresponding buffer solution, ATP and MnCl 2 into each DNA-TNA chimeric single chain in the same concentration and the same volume, enabling the CircLigase to catalyze the reaction of the DNA-TNA chimeric single chain in the buffer solution, and stopping the reaction after reacting at 55 ℃ for 12 h; finally, the fluorescence intensity of the residual single strand is measured by a denaturing polyacrylamide gel electrophoresis and fluorescence imaging system, and after the 2' -end of the 6 sequences TNA is identified to be added with 1 base, the cyclization yield is increased to be more than or equal to 70% after the cyclization yield reaction is 12 h.
  9. 9. The method for improving the cyclization yield of threose nucleic acid according to claim 1, wherein the step (4) is characterized in that an initial sequence and a corresponding final optimized sequence are selected, and the same concentration and the same volume are added into the CircLigase, a corresponding buffer, ATP and MnCl 2 , so that the CircLigase catalyzes a DNA-TNA chimeric single strand to react in the buffer, and the reaction is stopped after reacting at 55 ℃ for 2 h; and (3) determining the quantity of the residual single strands by denaturing polyacrylamide gel electrophoresis, calculating the cyclization yield of each sequence, comparing the change of the cyclization yields before and after optimization by drawing a histogram, and identifying that the cyclization yield of the final sequence 2h after all optimization is improved compared with the initial sequence after the final terminal base adding strategy optimization.
  10. 10. The method for improving the cyclization yield of threose nucleic acid according to claim 9, wherein the final optimized sequence is 8 sequences with the cyclization yield of 12h raised to 70% or more in the step (2) and 6 sequences optimized in the step (3).

Description

Method for improving cyclizing yield of threose nucleic acid Technical Field The invention belongs to the field of nucleic acid technology and non-natural nucleic acid application, and relates to a method for improving the cyclization yield of threose nucleic acid, in particular to a method for realizing that the cyclization yield of sequence 12h reaches more than 70% after 1G base is added at the 5 'end of sequence DNA and 1 or 2A bases are added at the 2' end of TNA. Background In recent years, cyclic nucleic acid has been widely focused in biological research due to its unique cyclic structure, which makes it free from the 5 'end and the 3' end of linear nucleic acid, thereby remarkably enhancing the degradation resistance to nuclease, having a longer half-life and higher stability in cells, and in addition, cyclic nucleic acid aptamer often exhibits good structural rigidity and highly specific target binding ability, and has important potential in the fields of drug delivery, targeted therapy, biosensing, and the like. Meanwhile, the circularized DNA template can also be used for rolling circle amplification, and further applied to disease diagnosis and biosensor development, and Threose Nucleic Acid (TNA) is an artificial nucleic acid formed by connecting alpha-L-threose through 2',3' -phosphodiester bonds, and has a skeleton structure simpler than DNA and RNA. TNA not only has excellent biostability, but also forms a stable double-stranded structure with DNA and RNA to realize genetic information exchange, so that TNA has been attracting attention in research of life origin and development of molecular tools. With the deep exploration of the functions, a plurality of TNA molecules which can target the aspergillin A, bind HIV reverse transcriptase, have RNA endonuclease activity and the like have been developed successively, and the application prospect is wide. In the prior art, a method for cyclizing TNA by using a CircLigase exists, but the main disadvantage of the method is that the cyclizing yield of partial sequences is low, and the potential of the method in the aspect of expanding the biotechnology field is not high. Disclosure of Invention The invention aims to provide a method for improving the cyclization yield of threose nucleic acid, which can greatly improve the cyclization yield of a sequence by adding G base at the 5 'end of the sequence DNA and adding A base at the 2' end of TNA. The technical scheme of the invention is that the method for improving the cyclization yield of the threose nucleic acid comprises the following steps: Step (1) taking a DNA 5 'end as a G base and a TNA 2' end as a base sequence as a substrate, observing the change of cyclization yield by changing the time and temperature of the cyclization reaction of the substrate, and exploring 12 h cyclization yields of 16 different terminal combined sequences, wherein the 16 sequences are named as initial sequences and are 4 multiplied by 4 combinations of a DNA 5 'end A, T, G, C base and a TNA 2' end A, T, G, C base; Wherein, only 2 initial sequences are 12h, the cyclization yield is more than or equal to 70 percent, the two combinations are DNA 5 'end is G base and TNA 2' end is A base sequence, and DNA 5 'end is G base and TNA 2' end is G base sequence; Step (2), for 14 different terminal base combination sequences with low cyclization yield, 1G base is added at the 5 'end of DNA and 1A base is added at the 2' end of TNA, and the influence of the strategy on the cyclization yield is studied; wherein, the cyclization yield of 8 sequences 12h is increased to be more than or equal to 70 percent, and the cyclization yield of 6 sequences is 40 to 60 percent; step (3) after adding 1A base at the TNA 2' end based on the step (2), optimizing the other 6 sequences with the cyclization yield of 40-60% again, so that the cyclization yield of the optimized sequences 12h is improved to be more than or equal to 70%; Step (4) comparing the cyclization yields of 14 initial sequences with lower cyclization yields with the cyclization yields of 2h of the corresponding final optimized sequences, wherein compared with the initial sequences, the cyclization yields of the optimized sequences 2h are obviously improved, and the maximum improvement is 30 times; The final optimized sequence is 8 sequences with the cyclization yield of 12 h in the step (2) being increased to more than or equal to 70% and 6 sequences optimized in the step (3), and the time point of 2h is selected because the reaction is in progress at the moment, and the improvement effect of the optimization strategy can be more accurately shown. Further, the step (1) is specifically that firstly, adding CircLigase, corresponding buffer solution, ATP and MnCl 2 to respectively modify monophosphate and Cy5.5 fluorophor in the 5' end and DNA, wherein the DNA-TNA chimeric single strand with G base at the 5' end and A base at the 2' end of the DNA is taken as a substrate, reacting f