The Construction And Application Of Nanomaterials Based On DNA Origami

DNA molecules have been used to build a variety of nanoscale structures anddevices over the past30years, DNA origami, which is based on the folding a longsingle-stranded virus DNA scaffold with the help of hundreds of short complementarystaple strands into numerous2D even3D well-defined shapes. Recently, DNAorigami structures have been widely investigated for various biomedical applicationssuch as diagnositics, therapeutics, biomimetics and so on.The emerging field of ‘DNAorigami’ has attracted the attention of many talented scientists in world, in efforts toexplore more simple design and synthesis method, to open up further areas ofapplication.My research mainly focused on developing a simple and generic method to formDNA origami nanostructures, exploring different properties and applications rangingfrom drug delivery, biosensor, nanodevice and DNA computing. Specifically asfollows:1. DNA Nanoribbons for Intracellular Delivery of Immunostimulatory DrugsDNA nanostructures have recently emerged as a type of drug deliverynanocarriers due to their suitable sizes, well-defined structures and low-toxicity. Here,we present a protocol for the assembly of DNA nanoribbon structures with rollingcircle amplification (RCA) and delivery of CpG oligonucleotide. DNA nanoribbonswith different dimensions and patterns were assembled with long RCA strands andseveral short staples. Significantly, we demonstrated they exhibited high-efficiencycellular uptake and improved immunostimulatory activity compared with ss-or ds-DNA. Interestingly, we recently have found these nanoribbons can also be assembledwithout RCA-amplified scaffold ssDNA. DNA nanoribbons with single, double ormultiple layers were successfully made with only four short synthetic strands. 2. The Label-Free Multiplexed Detection of Single-Molecules of MicroRNAsby Atomic Force Microscopy Based on the Hybridization Chain Reaction on DNAOrigamiMicroRNAs (miRNAs) are frequently deregulated in cancerous tissues and thusare regarded as potent cancer biomarkers for cancer classification and prognostication.Traditional methods, widely applied for detection of MicroRNAs, have an intrinsiclimitation on sample volumes, which are hard to scale down to single cell level. In thiswork, we proposed a label free, highly sensitive, and selective multiple miRNAsdetection method in single-molecule level based on the hybridization chain reactionon the origami with Atomic Force Microscopy (AFM). We demonstrated thesensitivity and specificity of the assay by detecting multiple MicroRNAs sequences.We found that the accurate control of the interprobe distances and anchorage lengthensures fast hybridization chain reaction kinetics. More importantly, the detectionassay is applied to identify the differential expression of miR21in extracted totalRNA samples of cancerous MCF-7cells and healthy breast cells. The results agreedvery well with previous reported analysis. This assay is of high potential forapplications in miRNAs expression profiling and early cancer diagnosis.3. A Dynamic DNA Nanodevice to Simulate The Falling Dominoes EffectDynamic DNA nanodevices play a key role in the field of DNA nanotechnologybecause they represent the prominent ability of human beings to control themechanical movement in molecular scale. Although many DNA nanodevices havebeen reported (such as switches, tweezers, rotaries, walkers, motors, etc.), they arestill in the early stages. The current DNA nanodevices are very primitive, and thecomplexity needs to be improved. To this end, we design and synthesize a dynamicDNA nanodevice, which successfully simulate dominoes falling as is well known. Thesquare DNA origami is used as a space to locate the dominoes, the hairpin DNAstrands H1of hybridization chain reaction system are employed as "dominoes", theDNA strand I trigger the falling of dominoes.With the aid of atomic force microscopewe can observe progressive collapse of the "dominoes" caused by trigger I. Comparedwith the dominoes game, these nanodevices based on hybridization chain reaction onDNA origami possess several superior features. Firstly, all the "dominoes" can beplaced in one step after setting the positions of "dominoes". Secondly, the "dominoes",which have been located on the origami, they are not disturbed by external factors.4. An Autonomous DNA Nanopaver for Maze Solving DNA computing could simplify the steps for solution because it took advantageof the many different molecules of DNA to try many different possibilities at once,which is fundamentally similar to parallel computing. Although DNA computing isfaster and smaller on certain specialized problems, it still needs many protocols doingthe experiments such as adding the strands step by step and obtaining the results fromthe gel. Here we applied a DNA nanorobot into DNA computing to simplify theprotocols in the experiment and obtained all the possibility of the resultssimultaneously under atomic force microscopy (AFM), we employed HCR(hybridization of chain reaction) system to create an automatic nanopaver which canpave on a typical track on a rectangular DNA origami. The simplest HCR systemcontains two hairpin species (H1and H2) and an initiator strand which works like acatalyst to trigger the chain reaction. Here, we selected the maze problem as a modelof DNA computing for our nanopaver to solve. Although the nanopaver could onlywalk in one path on each origami, all the paths would randomly disperse in thesolution that meant all the possibilities for the maze may read out by the AFM. Finally,we use magnetic bead separation to obtain the desired results.5. Site-Specific Immobilization of Antibodies in DNA Origami StructuresIgG antibodies, usually immobilized on various surfaces, have been extensivelyemployed in many fields such as purification of protein, medical diagnostics, andimmunosensors due to their extremely high specificity and strong binding ability.However, when antibody molecules are immobilized on a surface with randomorientation, their Fab region might be hidden, which results in the antibody–antigenbinding being hindered. Herein, we report a new method to attach an antibody in2Dorigami structures by preliminary non-covalent interactions and a subsequent covalentbinding. We constructed a2D DNA origami structure containing a cavity that fits theconstant domain of antibodies. On each side of the entry to this cavity will be placedtwo DNA strands containing tris-NTA nickel complexes for binding to the twohistidine clusters on the antibody.. Subsequently, covalent linkage to the antibody willbe induced by reaction of a lysine or a cysteine on the antibody to complementaryreactive esters or maleimides respectively on the DNA nanostructure.AFM imaging,agarose gel electrophoresis and binding assay have demonstrated that a combinationof noncovalent linkage and covalent linkage could afford a new class ofimmobilization method that would facilitate efficient loading of an antibody in a DNAnanostructure with control of the orientation.