Applications Of DNA Polyhedra In Cellular Imaging And Molecular Imprinting

Over the past few decades,DNA nanotechnology has evolved into a highly innovative and dynamic research area at the junction of chemistry science,biological science,materials science and nanotechnology.Based on Watson-Crick base-pairing principle and diverse self-assembly strategies,a variety of DNA nanostructures emerge in an endless stream,from simple to complex,from small to large,from one-dimension to three-dimension(3D).DNA polyhedral nanostructures are an important manifestation of this technology development,which inherits many unique advantages,including reliable structural rigidity,good biocompatibility,high programmability,accurate addressability,and rich functionalization approaches.They have played an important role in the fields of biosensing,drug delivery,logic computing,and materials fabrication.At present,in the field of biosensing,cellular imaging technology is in the ascendant,but still facing the problems of lack of stability,accuracy,real-time,and versatility;in the field of drug delivery,ideal drug delivery system should be able to protect drug molecules from degradation,increase solubility,reduce systemic toxicity or other biological side effects,penetrate the body barrier(upper epithelium and cell membrane),while the ability to target cells and controlled drug release are the points of concern in recent years;in the field of nanoparticle assembly,how to control the number and orientation of functional ligands on nanoparticles is a challenge that affects the diversity and physicochemical properties of self-assembled superstructure.In this thesis,combining with functional nucleic acid molecules and fluorescence detection technology,a variety of DNA polyhedron-based functional nanostructures have been designed for fluorescent imaging of nucleic acid and non-nucleic acid targets in living cells,as well as handling the number and position of DNA strands on the surface of gold nanoparticles.The detailed description is as follows:1.A DNA tetrahedron-based molecular beacon for tumor-related mRNA detection in living cellsAmong many DNA nanostructures,DNA tetrahedral nanostructures have attracted great attention in the field of bioanalysis and biomedicine due to their low cytotoxicity,good resistance to enzymatic cleavage and reliable cell membrane permeability.Molecular beacon(MB)probes have the advantages of high signal-to-noise ratio,high sensitivity and high specificity,and are commonly-used nucleic acid probes in biological analysis.However,MBs are difficult to penetrate the cell membrane and enter into the cellular environment without the aid of other carriers.To solve this problem,we developed a DNA tetrahedral nanostructures-based molecular beacon probe(DTMB).A hairpin structure was embedded in one edge of the tetrahedron,and two ends of the DNA strand complementary to the hairpin structure were respectively labeled with FAM fluorescent dye and Dabcyl quencher.In the presence of the target,the hairpin structure of DTMB changed from contraction to extension,inducing the spatial separation of fluorophore from the quencher to recover the fluorescent signal.In vitro fluorescence experiments and cell imaging experiments have demonstrated that DTMB could effectively detect tumor-related TK1 mRNA in living cells,and had good ability to distinguish different gene expression levels under drug incubation.2.Competition-mediated FRET-switching DNA tetrahedron molecularbeacon for intracellular molecular detectionIn the previous work,we reported a DNA tetrahedron-based molecular beacon(DTMB)that enables imaging of intracellular mRNA.Although it combines the advantages of molecular beacons,it still faces some problems.For example,the probe is not suitable for the detection of non-nucleic acids targets such as proteins and small molecules.Therefore,we constructed a competition-mediated fluorescence resonance energy transfer(FRET)switching DNA tetrahedral molecular beacon(CF-DTMB)for use as a universal detection platform in living cells.The optimization of this probe mainly included two points: first,introducing a competition-mediated chain displacement reaction to realize the separation of the recognition unit(aptamer)and the signal output unit;second,introducing a FRET signal output to eliminate the false positive signal and system disturbance.In the experiment,TK1 mRNA and adenosine triphosphate(ATP)molecules were used as model targets to achieve sensitive and accurate cellular imaging,which proved that CF-DTMB is a reliable tool for biosensing and disease diagnosis.3.Scallop-inspired DNA nanomachine: a ratiometric nanothermometer for intracellular temperature sensingTemperature is a very important type of physical and physiological indicators in biological and medical research.First,temperature is involved in the regulation of almost all chemical and biological reactions,such as gene expression,enzymatic reactions,energy conversion,and metabolism.Second,the vigorous proliferation of cancer cells will show abnormal temperature.Third,tumorous photothermal therapy requires accurate temperature feedback at the cell and tissue level.Therefore,the development of intracellular temperature sensing is of great significance.We developed a scallop-inspired DNA nanomachine(SDN)as a ratiometric nanometer thermometer for intracellular temperature imaging.By embedding a temperaturesensitive hairpin structure in the DNA tetrahedron,SDN can reversibly open and close based on the melting temperature(Tm),like a scallop.The experimental results showed that SDN not only had high temperature resolution and fast response,but also had good resistance to enzymatic cleavage and anti-interference.4.Three-dimensional molecular transfer from DNA nanocages to inner gold nanoparticlesDNA-modified functionalized gold nanoparticles have been an on-going research hotspot in the past few decades and are widely used in nanofabrication,optoelectronic devices,bioanalysis,disease diagnosis and cancer therapy.However,controlling the number and position of DNA on the surface of gold nanoparticles is a difficult challenge and has become one of the research focuses.The challenge is that the goldthiol covalent bonds are not selective and addressable under salt aging strategy.Thus,we used DNA icosahedral nanocage(I-Cage)to encapsulate gold nanoparticles in the lumen,and transferred the customized 3D DNA patterns to the surface of internal gold nanoparticles through the I-Cage template.Based on this strategy,a series of DNAfunctionalized gold nanoparticles(DPNPs)with different DNA numbers and positions were prepared.Importantly,DPNPs inherited the DNA pattern transferred from the template with high fidelity,completely retaining sequence specificity.This method is a combination of nanotechnology and ‘lithography' technology,and is expected to play a role in the field of nanomaterial assembly and accurate disease diagnosis.