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Method for regulating and controlling self-assembly of nucleic acid nanostructure based on DNA stem-loop conformation transformation

A nucleic acid nanometer and nanostructure technology, applied in the field of DNA nanometers, can solve problems such as restrictions on wider application and lack, and achieve the effect of flexible regulation

Pending Publication Date: 2020-04-17
苏州朴衡科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing self-assembly of nucleic acid nanostructures mainly has the following problems: I. In terms of structure formation, it mainly relies on thermal annealing with gradient temperature changes, which limits its wider application in vivo
II. In terms of dynamic regulation, there is a lack of modular switches to fine-tune the self-assembly process, thereby generating a deeper understanding of the mechanism of action

Method used

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  • Method for regulating and controlling self-assembly of nucleic acid nanostructure based on DNA stem-loop conformation transformation
  • Method for regulating and controlling self-assembly of nucleic acid nanostructure based on DNA stem-loop conformation transformation
  • Method for regulating and controlling self-assembly of nucleic acid nanostructure based on DNA stem-loop conformation transformation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] (1) Design the sequences of DNA strands A, B, C, and I required for assembling the Y-shaped structure, wherein the 5' to 3' ends of the sequence A are successively a, s, b, c*, b* regions, at 5 'modified FAM fluorescent group; the 5' to 3' of sequence B are b, c, a*, c* regions in sequence; the 5' to 3' ends of sequence C are c, a, b*, a* regions in sequence, Dabcyl quenching group is modified at 3'; the 5' to 3' ends of sequence I are sequentially b*, t, and a* regions; wherein a, b, and c regions are complementary to the sequences of a*, b*, and c* regions, respectively For pairing, the S region is the stem-loop region, and the main region is inserted into a 6nt protection region to stabilize the key structure in the reaction pathway.

[0066] (2) Design the sequences of the stems of the stem-loop structure in chain A, the lengths of which are 4, 5, 6, 7, and 8 nt, respectively.

[0067] (3) At 25°C, the nucleic acid sequences A, B, C, and I were added to TE / Mg in a ...

Embodiment 2

[0069] (1) At 25°C, the A chain in the S-Off state and the chains B, C, and I were mixed in TE / Mg at a molar ratio of 1:1.2:1.5:1 2+ The buffer system is mixed well. At this time, because the switch has a rigid double-chain structure, which "separates" the branch migration end from the toehold end, the chain replacement reaction is difficult to proceed, so no Y-shaped structure is formed.

[0070] (2) Add 2.5nM, 5nM, 6nM, 7.5nM, 10nM and 40nM activated strands to the above buffer solution respectively. Due to the chain displacement reaction between the activated strand and the inhibitory strand, a stem-loop structure is formed, and the branch migration end and toehold The end distance is shortened, the hairpin A structure is opened by chain I, and then B, C and A pair with each other to form a Y-shaped nanostructure of A-B-C. The minimum concentration of the activated chain added was 2.5nM (0.1x), which indicated that the conformational transformation of the stem-loop switch w...

Embodiment 3

[0072] Because a stem-loop switch can only regulate whether the Y-shaped nanostructure is formed, but cannot finely regulate the intermediate assembly process. In this experiment, three stem-loop switches with different sequences were used to implement multi-step controllability on Y-shaped nanostructures.

[0073] (1) Sequence design

[0074] Add a stem-loop switch to the toehold and branch migration regions of the A, B, and C strands respectively, and design the sequences of three stem-loop structures, in which the length of the stem is 7nt, and the loop is 15nt; design the inhibitory chain and the activation chain corresponding to the stem-loop , and its toehold length is 7nt.

[0075] (2) Step-by-step regulation

[0076] At 25°C, the A, B, C chains and I chains in the S-Off state were mixed in TE / Mg at a molar ratio of 1:1.2:1.5:1. 2+ Mix the buffer system. Since there are three stem-loop switches in the system, different structures can be produced by adding different ...

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Abstract

The invention discloses a method for regulating and controlling self-assembly of a nucleic acid nanostructure based on DNA stem-loop conformation transformation. A stem-loop switch with a variable structure is inserted between a sticky toehold region and a branch migration region; when the switch is in an S-ON state, the switch has a stem-loop structure, the toehold end and a branch migration endare affected little, so that a chain replacement reaction is performed successfully and thus a nano structure is formed through assembling; when an inhibition chain is added, a loop region and the inhibition chain are complementarily paired to form a rigid double-chain structure, the switch is changed into an S-OFF state, a branch migration end and the toehold end are separated, occurrence of a chain replacement reaction is inhibited to prevent assembling of a next step. Since the S-ON state and the S-OFF state can be freely switched in a reaction system, the self-assembly process of the Y type nucleic acid nanostructure can be flexibly regulated and controlled in real time at the constant temperature, so that the application potential of the nucleic acid nanostructure in the fields of biosensing, biological detection, drug delivery, disease treatment and the like is further expanded.

Description

technical field [0001] The invention belongs to the field of DNA nanotechnology, and in particular relates to a method for regulating the self-assembly of nucleic acid nanostructures based on DNA stem-loop conformation transformation. Background technique [0002] The DNA molecule is not only a carrier for organisms to store and transmit genetic information, but also a universal element for manufacturing nanoscale components and devices. Utilizing the predictability and programmability of Watson Crick base pairing, a bottom-up method is used to construct nucleic acid nanostructures with good biocompatibility and stability, and to be used in biosensing and biodetection , Drug delivery, disease treatment and other fields have shown important application potential. [0003] The DNA stem-loop is a special structure capable of conformational transformation: both ends contain a stem of a specific length, and a loop structure in the middle. In the free state, the stem region spec...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/10C12Q2531/119
Inventor 裴昊俞慧珍李丽
Owner 苏州朴衡科技有限公司
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