摘要
DNA纳米结构作为理想的纳米支架,具有空间可寻址、几何结构可编程、易于化学修饰、生物相容性好等优势,现已广泛应用于实现各类生物分子在纳米尺度的定向排列与组装,并逐渐在多个交叉领域发挥重大作用.DNA折纸术的出现更是提供了结构更为复杂且精确的DNA骨架设计.本文首先从寡核苷酸链与生物分子的偶联、DNA-生物分子复合物的组装与纯化这三个方面简述了DNA骨架与生物分子的基本组装方法.随后,着重总结了DNA纳米结构-生物大分子自组装体在酶级联反应体系构建中的应用以及DNA纳米支架在AFM、荧光以及冷冻电子显微镜等辅助生物成像技术方面的应用.最后,综合讨论了DNA纳米技术本身在以上领域中目前存在不足并提出展望.
Abstract
Arranging biomacromolecules in a specific order and orientation on the nanoscale has long been a prominent research topic.Significant progress has been made in understanding intermolecular interactions within bionetworks and elucidating biosystem mechanisms.DNA nanostructures,serving as ideal scaffolds,have been extensively employed to achieve precise arrangement of diverse biomolecules at the nanoscale due to their well-defined geometry,programmability,addressability at the nanometer scale,functionalization capabilities and excellent biocompatibility.The advent of DNA origami has further enhanced the ability to design intricate and accurate DNA frameworks.This technique utilizes numerous short DNA strands,called staples,to fold a longer DNA strand,known as the scaffold,enabling the creation of more structurally complex and precise DNA architectures.This review is mainly structured into four main sections.Firstly,we provide a comprehensive overview of the various methods employed for the three key steps involved in assembling DNA nanostructures with biomolecules.These steps encompass the conjugation of oligonucleotides and biomolecules,the hybridization of DNA nanostructures with biomolecules,and the purification of DNA nanostructures-biomolecule complexes.Secondly,we briefly summarize the relevant applications of DNA nanostructures-biomolecules self-assembly,with a primary focus on the construction of enzyme cascade reaction systems.The combination of enzyme cascades and addressable DNA assembly has proven highly effective in creating stable artificial multi-enzyme complexes.This integration allows for precise control over the position,shape,and composition of the complexes,enabling efficient manipulation of enzyme distance,substrate channeling,and compartmentalization.Thirdly,we focus on the application of DNA nanostructures in diverse imaging techniques,including atomic force microscopy(AFM),fluorescence imaging,and cryo-electron microscopy(cryo-EM).DNA nanostructure offers distinct advantages in advancing bioimaging modalities,such as enabling multiplexed imaging,facilitating logical responsive imaging,and enabling protein structural analysis.While AFM has been extensively employed for the characterization of various biomolecules,it still has limitations in direct observation and comparison of freely dispersed molecules.However,by utilizing DNA nanostructures as a platform for loading molecules,it becomes possible to realize the visualization of dynamic intermolecular interactions using AFM.Besides,through precisely manipulating the number and combination of selected fluorophores,DNA nanostructures provide a means to mitigate the issue of spectral overlap among fluorophore colors.This precise control allows for tuning the overall color of probes,effectively overcoming spectrum overlapping and enabling the generation of distinct and distinguishable colors.Moreover,DNA nanostructures show potential in assisting the determination of protein orientation during cryo-electron microscopy(cryo-EM)imaging and potentially mitigating issues such as protein unfolding or nonrandom absorption at the water-air interface.The final section of this review discusses the technical challenges that need to be addressed to enhance the utilization of DNA nanoscaffolds,for example achieving precise orientation control of biomolecules on DNA scaffolds,optimizing the stability of the overall complexes to achieve in vivo functionality and overcoming technological bottlenecks that impede the application of DNA nanoscaffolds in cryo-EM imaging.
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