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CN-121975663-A - Bacterial surface modification method based on programmable pilus DNA nanowire stick and application

CN121975663ACN 121975663 ACN121975663 ACN 121975663ACN-121975663-A

Abstract

The application discloses a bacterial surface modification method based on a programmable pilus DNA nanowire and application thereof. The method comprises the steps of firstly constructing a DNA nanowire bar structure with a three-dimensional structure by using a DNA paper folding technology, respectively modifying functional molecules on the head end and the main body of the DNA nanowire bar structure, and incubating bacteria modified with coupling groups and the DNA nanowire bar structure modified with the functional molecules. The DNA nanowire bar structure constructed by the application can be used for regulating and controlling the movement of bacteria, and the behavior of the bacteria can be regulated regularly without genetic manipulation by regulating the geometric shape and the density of the pilus analogue based on DNA. The DNA nano-wire rod has the advantages of modifiable property, low toxicity and low immunogenicity, can be used for analyzing how the nano-grade appendage morphology controls the movement, and lays a foundation for the application in the interface adaptability, field planting or engineering probiotic systems in the future.

Inventors

  • WANG XIULI
  • HUANG KUI
  • ZHANG MENGMENG
  • ZHU YUFENG
  • LIU JINYAO
  • YANG YANG

Assignees

  • 上海市浦东新区浦南医院(上海交通大学医学院附属仁济医院浦南分院)
  • 上海交通大学医学院附属仁济医院

Dates

Publication Date
20260505
Application Date
20260330

Claims (10)

  1. 1. A method for modifying the surface of bacteria based on programmable pilus DNA nanowires, the method comprising the steps of: Firstly, constructing a DNA nanowire or nanorod structure with a three-dimensional structure by using a DNA paper folding technology; Step 2, respectively modifying functional molecules on the head end and the main body of the DNA nanowire or nanorod structure; And 3, performing a coupling reaction on the bacteria modified with the coupling molecules or groups and the DNA nanowire or nanorod structure modified with the functional molecules to enable the DNA nanowire or nanorod to be grafted on the surface of the bacteria, wherein the coupling molecules or groups are molecules or groups capable of being combined with the functional molecules.
  2. 2. The method of claim 1, wherein the modification of the functional molecule is by hybridization of the complementary sequence of the modified functional molecule to an anchor sequence extending from a selected site of the DNA nanowire or nanorod structure using the principle of DNA base complementary pairing, thereby modifying the functional molecule to the head and body sites of the DNA nanowire or nanorod structure, respectively.
  3. 3. The method according to claim 1, wherein the method for constructing the DNA nanowire or nanorod structure comprises: A1, constructing a DNA nanowire or nanorod structure, presenting a structural schematic diagram by using a 3D image, and then designing the DNA nanowire or nanorod structure by using DNA nanostructure professional design software caDNAno; step A2, further constructing variants of the DNA nanowire or nanorod structure on the basis of the step 1, and establishing modification sites on the head end and the main body; and A3, performing structural assembly and purification on the DNA nanowire or nanorod structure constructed in the step 2.
  4. 4. The method of claim 3, wherein the step 1 further comprises obtaining the corresponding sequences of hundreds of staple chains by inputting the sequences of the template chains, and performing sequence synthesis, wherein the long-chain ssDNA template p7560 in the genome of the M13 phage is used as the template chain sequence of the DNA nanowire structure, and the long-chain ssDNA template p3024 in the genome of the M13 phage is used as the template chain sequence of the DNA nanorod structure.
  5. 5. The method according to claim 4, wherein the self-assembly and purification of the structure in the step 3 specifically comprises annealing the template strand sequence and the synthesized staple strand sequence, performing base pairing self-assembly to obtain a DNA nanowire or nanorod, and separating and purifying to obtain a purified DNA nanowire or nanorod, wherein the purification comprises obtaining a purified structural monomer by using an ultracentrifugation method and an ultrafiltration concentration method, and removing unbound staple strands and dimer and multimer impurities.
  6. 6. The method of claim 1, wherein the coupling molecule or group is covalently bound to the functional molecule in step 3 via a chemical bond, the chemical bond being an amide bond, a thiol-to-thiol bond, or a biotin-avidin bond, the functional molecule further comprising one or more of a fluorescent molecule, a nucleic acid, and a biotin-streptavidin protein.
  7. 7. The method according to claim 1, wherein the step 3 specifically comprises the steps of reacting bacteria with Sulfo-NHS-Biotin to modify Biotin on the surface of the bacteria, then coupling the bacteria with streptavidin to obtain bacteria modified with Biotin-streptavidin, and then coupling the bacteria modified with Biotin-streptavidin with DNA nanowires or nanorod structures modified with Biotin to graft the DNA nanowires or nanorods on the surface of the bacteria.
  8. 8. The method of claim 1, wherein the concentration of the bacterial surface grafted DNA nanowires or nanorods is 0.5 to 40nm.
  9. 9. A bacterium obtained by the method according to any one of claims 1 to 5.
  10. 10. Use of the bacteria obtained by the method according to any one of claims 1 to 5 as a tool strain for studying bacterial biological behaviour and mechanism of action.

Description

Bacterial surface modification method based on programmable pilus DNA nanowire stick and application Technical Field The application relates to a bacterial surface modification method and application based on a programmable pilus DNA nanowire, belonging to the technical fields of DNA nanotechnology and microbial engineering. Background In 1982, seeman et al first proposed rules for constructing DNA branching junctions using Watson-Crick base pairing, opening a new era of DNA nanotechnology. Over the next few decades, DNA has been widely accepted and applied as a versatile polymeric material for the construction of more complex micro-nanostructures in the world of matter. In 2006, paul Rothemund reported DNA paper folding for the first time, and the DNA paper folding technology further promoted the research and application of DNA nanotechnology. Based on the advantages of good biocompatibility, precise surface multiposition modification, controllable size and shape and the like of the DNA nano structure, the nano structure obtained by DNA self-assembly is approximately level with the substructures of cells and bacterial appendages in the aspects of dimension, morphology, rigidity and the like, has extremely strong comparability, and is a leading edge means for constructing a bionic model system. For example, the work of using DNA assembly framework structures to mimic transmembrane pore channels, cytoskeletal proteins, nuclear pore complexes, eukaryotic viruses and phages has progressed significantly in recent years. Bacteria interact with the environment through a variety of surface attachment structures, with pili being one of the most versatile and evolutionarily conserved structures. These filamentous organelles are only a few nanometers in diameter and can be several micrometers in length, mediating critical actions such as surface adhesion, twitch movement, biofilm formation, and horizontal gene transfer through dynamic stretch-shrink cycles. Mechanical action of type IV pili, for example, can generate up to 100pN of propulsion, driving the bacteria in a restricted or high viscosity environment. Such movement and adhesion processes are critical for bacterial colonization, pathogenicity and environmental adaptation. Manual regulation of bacterial movement has become an emerging topic in the area of intersection of microbiology and material science. Current research mainly uses synthetic coatings of polymers, nanoparticles, and peptide conjugates to alter bacterial adhesion, biofilm formation, or population dynamics, but these chemical strategies often lack structural precision, resulting in uneven surface modification and unpredictable mechanical properties. Although micro-processing environment or external field can influence the motion trail of bacteria, the nano-scale structural characteristics of natural fimbriae can not be reproduced. Pili are both virulence factors for pathogens and adhesion mediators for probiotics, and play a role in both infection and beneficial colonization. Although biochemical and structural studies have elucidated pilin composition and assembly pathways, direct manipulation of the geometric characteristics of the pili (e.g., length, diameter, or mechanical flexibility) remains a technical bottleneck. The traditional gene knockout or overexpression system can only change the bacterium Mao Fengdu, and is difficult to accurately regulate the nanoscale structure or biomechanical response. Thus, there remains a long standing challenge to develop a controllable, quantitative method of simulating and modulating pilus-like structures on living bacteria, whereby the system studies how the geometry modulates the ability to exercise. In recent years, the rapid development of DNA nanotechnology makes it possible to realize precise structural design and synthesis on the micro-nano scale. Meanwhile, cross-fusion of the nucleic acid nanotechnology field with biomedical science is receiving increasingly widespread attention. Although numerous nanomaterials have been reported to be modifiable to bacterial surfaces, studies of DNA nanostructures for bacterial modification are still in the beginning and related reports are very limited. Therefore, how to adopt DNA strategies to realize controllable modification of bacterial surfaces, improve the bioavailability of bacterial preparations and regulate the movement behavior of bacteria is a current urgent problem to be solved. Disclosure of Invention The application aims to provide a simple and effective novel strategy for controllably modifying the surface of bacteria by designing a simulated pilus DNA bar structure and introducing the advantages of controllable shape, controllable length and multi-site modification to the surface of bacteria by utilizing a DNA paper folding technology, and the novel strategy can be used as an engineering bacteria for researching the influence mechanism of pili on the motion behavior change of bacteria, thereby