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Preparation method for circular single-stranded DNA integrated with aptamer and applications of circular single-stranded DNA integrated with aptamer in DNA origami

A nucleic acid aptamer and aptamer technology, applied in the fields of DNA nanotechnology and biological applications, can solve the problems of low ligation efficiency and stability, and achieve the effects of efficient preparation and rich diversity

Pending Publication Date: 2021-08-20
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above problems, the present invention provides a preparation method of circular single-stranded DNA integrated with nucleic acid aptamers, and constructs it into a functional DNA origami structure, which breaks through the low connection efficiency and stability of DNA origami nanostructure functionalization The problem

Method used

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  • Preparation method for circular single-stranded DNA integrated with aptamer and applications of circular single-stranded DNA integrated with aptamer in DNA origami
  • Preparation method for circular single-stranded DNA integrated with aptamer and applications of circular single-stranded DNA integrated with aptamer in DNA origami
  • Preparation method for circular single-stranded DNA integrated with aptamer and applications of circular single-stranded DNA integrated with aptamer in DNA origami

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preparation example Construction

[0028] As shown in the figure; a method for preparing a circular single-stranded DNA integrated with a nucleic acid aptamer, and using the obtained circular single-stranded DNA integrated with a nucleic acid aptamer to construct a functional DNA origami structure, the specific steps are as follows:

[0029] (1.1), constructing circular double-stranded recombinant phagemids containing nucleic acid aptamer sequences;

[0030] (1.2), transforming the constructed circular double-stranded recombinant phagemid into Escherichia coli competent cells; extracting circular single-stranded DNA with nucleic acid aptamer integration.

[0031] Further, in the step (1.1), the specific operation method for constructing a circular double-stranded recombinant phagemid containing a nucleic acid aptamer sequence is as follows:

[0032] First, the selected nucleic acid adapter fragments are obtained by using chemical synthesis, and the nucleic acid adapter fragments are connected to the DNA fragmen...

Embodiment 1

[0043] Preparation of ssDNA integrated with human α-thrombin aptamer TBA-15 and TBA-29 and PDGF aptamer;

[0044] Utilizing the present invention, the preparation of human α-thrombin aptamer TBA-15 and ssDNA integrating TBA-29 and PDGF aptamer comprises the following steps:

[0045] (1), constructing a circular double-stranded recombinant phagemid containing a nucleic acid aptamer sequence;

[0046] Human α-thrombin aptamers TBA-15 and TBA-29 and PDGF aptamer fragments were obtained by chemical synthesis; two human α-thrombin aptamers TBA-15 and TBA-29 were synthesized by overlapping PCR. The PDGF aptamer fragment (sequence shown in Table 1) was connected to the DNA fragment of the selected region in the M13mp18 RF DNA vector; the product of overlapping PCR was purified by agarose gel electrophoresis and then assembled by Gibson The method was inserted into the M13mp18 RF DNA vector and replaced the original DNA fragment in this region, and verified by gene sequencing, the re...

Embodiment 2

[0052] Example 2. Constructing a functional DNA origami structure of nucleic acid aptamers integrating backbone chains:

[0053] Using the ssDNA integrated with the nucleic acid aptamer prepared by the present invention to construct a functional DNA origami structure, the specific operations are as follows:

[0054] Mix the circular ssDNA integrated with different aptamers prepared in Example 1 with their corresponding staple strands at a molar ratio of 1:10; according to the requirements of subsequent experiments, the final concentration of ssDNA is in the range of 1-10 nM; add A certain amount of 1×TAE / Mg 2+ Buffer (Mg 2+ The concentration is 12.5mM) to 100μL and mixed well, then placed in the PCR instrument, set the program gradient annealing from 85°C to 25°C for 16h; Purified by agarose gel electrophoresis.

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Abstract

The invention discloses a preparation method for circular single-stranded DNA integrated with aptamer and applications of the circular single-stranded DNA integrated with aptamer in DNA origami, and belongs to the field of DNA nanotechnologies and biological application. The preparation method mainly includes the following steps: (1.1) constructing circular double-stranded recombinant phagemid including an aptamer sequence; and (1.2) converting the constructed circular double-stranded recombinant phagemid into competent escherichia coli cells; and extracting the circular single-stranded DNA integrated with aptamer. Through the preparation method, a user defined skeleton chain sequence can be designed and produced, so as to add various function sequences at a user defined position. Problems of poor efficiency and stability of structural functionalization of traditional DNA origami can be broken through.

Description

technical field [0001] The invention belongs to the field of DNA nanotechnology and biological applications, in particular to a method for preparing circular single-stranded DNA integrated with nucleic acid aptamers and its application in DNA origami; in particular, it relates to the preparation of different nucleic acid aptamers Functionalized single-stranded DNA (single-stranded DNA, ssDNA) is used as the backbone strand of DNA origami to construct functional DNA origami structures. Background technique [0002] As a natural biomacromolecule, DNA has unique advantages in the construction of functional nanostructures due to its unique chemical structure and unique intermolecular interactions; DNA molecules can self-assemble to form nanostructures with complementary base pairing. Ordered structure with nanoscale precision; this DNA nanostructure can also modify the DNA chain at a specific position, making it a scaffold to guide other molecules or nanomaterials to perform con...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/70C12R1/19
CPCC12N15/10C12N15/70C12Q2531/113
Inventor 李喆史野陈小星汪倩于涵洋
Owner NANJING UNIV
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