Method for assisting in sorting lipidosome by utilizing single-stranded DNA nanostructure

A nanostructure and liposome technology, which is applied in liposome delivery, pharmaceutical formulations, medical preparations of non-active ingredients, etc., can solve the problem of uneven product, achieve high uptake rate, high cost, and good stability sexual effect

Active Publication Date: 2020-11-03
FUDAN UNIV SHANGHAI CANCER CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A second strategy is to remodel the membrane structure of liposomes by on-demand oligomerization or reconfiguration of the DNA apparatus, whic

Method used

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  • Method for assisting in sorting lipidosome by utilizing single-stranded DNA nanostructure
  • Method for assisting in sorting lipidosome by utilizing single-stranded DNA nanostructure
  • Method for assisting in sorting lipidosome by utilizing single-stranded DNA nanostructure

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Embodiment 1

[0041] The sequence design of embodiment 1 single-stranded DNA nanostructure

[0042] According to the design principle of the PX structure, taking a triangle as an example, two parallel double helices in each side are connected to another chain by crossing at a point close to each other. This structure can be formed by assembling a single-stranded DNA. The length of PX is designed to be between 10 and 20 nm on each side, so that its morphology can be clearly seen under the atomic force microscope. Then three PX molecules are covalently linked head-to-tail by 5'-3' to 5'-3' linkages to form triangles to ensure that the nanostructures to be generated can self-assemble from long ssDNA with a length of approximately 520 nt (e.g. figure 1 ), its sequence is shown in SEQ ID NO:1 sequence. Several TN (N=5 or 7) loops were placed at some tips to relax constraints in the edge-edge connection process.

Embodiment 2

[0043] Example 2 Construction of phagemids comprising complete DNA nanostructure sequences and deoxyribozyme cleavage substrates

[0044] The complete sequence inserted into the phagemid vector consists of two parts: the deoxyribonase recognition site and the nanostructure sequence site. The nanostructure sequence is arranged in the middle, and the DNAzyme recognition sites are on both sides. Randomly select about 10 to 20 nucleotides as a spacer sequence and distribute it between the DNAzyme and the site of the DNA nanostructure to avoid steric hindrance between the two, thus ensuring the former in ssDNA amplification Even in the case of large structures in the vicinity, the daughter normally forms a hairpin structure and thus cuts normally. Taking the triangular structure as an example, a highly active self-cleaving DNAzyme (I-R1a) with a length of 45 nt (sequence: 5'-GATGTACAGCCATAGTTGAGCATTAAGTTGA / AGTGGCTGTACATC-3') was buried on both sides of the nanostructure site. Whe...

Embodiment 3

[0045] Example 3 Helper Phage Assisted Amplification of Single-Stranded DNA

[0046] A single p3024-triangle transformed colony was grown overnight at 37°C in 20 ml LB medium containing ampicillin (100 μg / mL). 2x YT medium (16.0g / L trypsin, 10.0g / L yeast extract, 5.0g / L NaCl, 5mM MgCl 2 ) were inoculated with 3 mL of overnight culture, and shaken at a speed of 250 r / min. When the OD600 of the culture reached about 0.4-0.5, VCSM13 helper phage was added thereto to an MOI of 20. After 30 min, kanamycin (final concentration 50 μg / mL) was added to the culture to select infected cells. After about 4.5 hours, the culture was collected and centrifuged at 4000 rcf for 15 minutes at 4°C to remove bacteria. The supernatant was transferred to a clean bottle and dissolved with PEG 8000 (40 g / L) and NaCl (30 g / L). The mixture was incubated on ice for 30 minutes and then centrifuged at 5000 rcf for 30 minutes at 4°C. The supernatant was discarded and the phagemid pellet was resuspended ...

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Abstract

The invention discloses a method for assisting in sorting lipidosome by utilizing a single-stranded DNA nanostructure. The method comprises the following steps of connecting a cholesterol-modified single-stranded DNA nanostructure with lipidosome to form a lipidosome complex, centrifugally separating out different components through iodixanol gradient density, and finally, obtaining a lipidosome library with uniform size in different components. Besides, the invention also provides the liposome complex, and the liposome complex can be used for effectively sorting liposomes with different sizes. An ssDNs auxiliary sorting technology easily produces the lipidosome with the average diameter of about 40-140nm, so that an attractive platform is provided for systematically exploring the size andthe shape of the lipidosome coated by ssDNs and related to absorption of the lipidosome by living cells.

Description

technical field [0001] The invention belongs to the technical field of liposome regulation and relates to a method for assisting sorting liposomes by using single-stranded DNA nanostructures. Background technique [0002] DNA origami (DNA origami) is a mainstream method of DNA nano-self-assembly. Usually, a DNA main chain is assisted by hundreds or thousands of synthetic DNA short chains, which are folded and locked according to the principle of complementary base pairing to generate designed structures. Nano-structure. Single-stranded DNA origami (ssDNA origami) is an evolution and derivative of traditional DNA origami, which eliminates the need for many short sequences in traditional origami, and integrates sequence information into a long single In stranded DNA, complex and controllable nanostructures can be self-assembled from a single DNA sequence. Compared with multi-strand DNA origami, single-strand DNA origami avoids the defects and errors that may be formed during...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K47/26A61K47/28A61K47/22A61P35/00
CPCA61K9/1277A61K9/1271A61K47/26A61K47/28A61K47/22A61P35/00
Inventor 顾宏周陈黎曼
Owner FUDAN UNIV SHANGHAI CANCER CENT
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