Construction method and application of aptamer-DNA macromolecular polymer based on non-linear hybridization chain amplification

A high molecular polymer, hybrid chain technology, applied in the construction and application of DNA high molecular polymers, can solve the problems of low bioavailability, strong side effects, non-targeting, etc., and achieve the effect of increasing application potential

Active Publication Date: 2019-02-01
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional chemotherapeutic drugs, such as doxorubicin and paclitaxel, are limited in practical application due to shortcomings such as no targeting, strong side effects, and low bioavailability.

Method used

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  • Construction method and application of aptamer-DNA macromolecular polymer based on non-linear hybridization chain amplification
  • Construction method and application of aptamer-DNA macromolecular polymer based on non-linear hybridization chain amplification
  • Construction method and application of aptamer-DNA macromolecular polymer based on non-linear hybridization chain amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Synthesis of DNA polymers based on nonlinear HCR

[0044] (1) After dissolving the substrate strands S1 and S3, use a micro-ultraviolet spectrophotometer Q5000 to accurately measure the concentration, treat with 1uL phosphorylase, react in a PCR instrument at 37°C for 3h, and then treat at 95°C for 10min to end the phosphorylation process. Obtain base strands S1* and S3*;

[0045] (2) The bottom strands S1* and S2, and the bottom strands S3* and S4 were mixed at a molar ratio of 1:2, annealed in a PCR instrument at 90°C for 10 minutes, and cooled to 0.1°C / s to At 25°C, the main chain S1*S2 and the main chain S3*S4 were obtained, and stored at 4°C for later use;

[0046] (3) Auxiliary chains H1 and H2 are added to the solution of main chain S1*S2 and main chain S3*S4 respectively, the molar ratio of auxiliary chain H1 to auxiliary chain H2 is 1:2, and the ratio of auxiliary chain H1 to main chain S1*S2 The molar ratio is 1:3~2:3, and incubated in a PCR instru...

Embodiment 2

[0051] (1) After dissolving the base strands S1 and S3, use a micro-ultraviolet spectrophotometer Q5000 to accurately measure the concentration, heat in a PCR instrument at 37°C for 3 hours, and then treat at 95°C for 10 minutes to obtain the base strands S1* and S3*;

[0052] (2) The bottom strands S1* and S2, and the bottom strands S3* and S4 were mixed at a molar ratio of 1:2, annealed in a PCR instrument at 90°C for 10 minutes, and cooled to 0.1°C / s to At 25°C, the main chain S1*S2 and the main chain S3*S4 were obtained, and stored at 4°C for later use;

[0053] (3) Auxiliary chains H1 and H2 are added to the solution of main chain S1*S2 and main chain S3*S4 respectively, the molar ratio of auxiliary chain H1 to auxiliary chain H2 is 1:2, and the ratio of auxiliary chain H1 to main chain S1*S2 The molar ratio is 1:3~2:3, and incubated in a PCR instrument at 37°C for 30min;

[0054] (4) Mix the two solutions in step (3) evenly, add the initiator chain, the amount of the in...

Embodiment 3

[0058] The gap-unclosed DNA polymer obtained in Example 1 and the gap-closed DNA polymer obtained in Example 2 were respectively reacted in a PCR instrument at 37°C for 0-36h, followed by polyacrylamide gelation. Gel electrophoresis detection, see the results figure 2 , figure 2 It is the measurement diagram of serum stability and temperature stability of two different DNA polymers (A: DNA polymer with open gap; B: DNA polymer with gap closed). It can be seen from the figure that the unlinked DNA polymer was degraded to a certain extent after 12 hours of serum incubation, and the degree of degradation gradually increased with the extension of incubation time, and most of the samples blocked in the gel pores had been degraded at 36 hours . In contrast, the linked DNA polymer has almost no effect, even if the incubation time reaches 36h, it still cannot degrade it.

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Abstract

The invention discloses construction and application of a DNA macromolecular polymer based on non-linear hybridization chain amplification. An initiating chain is introduced in the system, and six different non-hairpin nucleic acid probes are induced to perform cascading self-assembly to form the DNA macromolecular polymer through a non-linear hybridization chain reaction. On the basis, phosphorylase is used for treating a 5' end of a part of assembly sequence, and a DNA assembly with higher stability is formed under the action of ligase. Nucleic acid aptamers are combined at a viscous tail end to perform targeted recognition of target cells and are introduced into a cytotoxic drug doxorubicin, and imaging and drug administration of the target cells are achieved.

Description

technical field [0001] The invention belongs to the technical field of biomedical targeted drug delivery system, and in particular relates to the construction and application of a DNA polymer based on nonlinear hybrid chain amplification. Background technique [0002] At present, in vitro nucleic acid amplification signal amplification technology to improve detection sensitivity and accuracy has become a hot research direction in the field of molecular biology. Common nucleic acid isothermal signal amplification techniques can be roughly divided into: strand displacement amplification (Strand displacement amplification, SDA), rolling circle amplification (rolling circle amplification, RCA), hybridization chain reaction (Hybridization chain reaction, HCR), Loop-Mediated Isothermal Amplification (LAMP) and so on. HCR technology is a new type of nucleic acid isothermal amplification technology that does not involve enzymes in catalysis and constant temperature. Under certain c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34A61K47/26A61K31/704A61P35/00
CPCA61K47/26A61P35/00A61K31/704C12P19/34
Inventor 邵敬伟乐景青徐建国
Owner FUZHOU UNIV
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