Self-assembly Of Functional DNA Nanoconstruction For Biomedical Applications

Since Waston and Crick’s found Watson Crick base pairing and a well characterized double-helical structure in 1953, molecular biology has been developed rapidly. Compared with traditional inorganic nanomaterials, DNA has been increasingly emerging as powerful and versatile nanoscale building blocks for the construction of precisely predefined nanostructures by its unique properties such as desirable molecular recognition via highly specific Waston Crick base pairing, ease of chemical functionalization and spatial addressability. Sequence-design principles for programming nucleic acids to self-assemble into stable, highly structured macromolecular assemblies date back to foundational work by Ned Seeman of New York University in the early 1980 s. During the past three decades, the ingenious construction of two- and three-dimensional objects with desired shapes and geometries using the “bottom-up” DNA selfassembly has significantly advanced, which provides promising approaches for building highly-ordered nanostructure materials with nanometer-scale addressability. Despite that the assemblies of highly structured macromolecules by different DNA building blocks have been extensively explored, little attention has been paid to employ such assemblies as potential signal amplifiers to transduce molecular recognition events.The development of simple and cost-effective DNA nanostructure with new functionality will therefore facilitate the construction of different nanoconstruction for various applications. In response, we report here the construction of a DNA nanostructure and the application of this nanostructure for simple and sensitive monitoring of different nucleic acid biomarkers for early diagnosis of diseases. Part 1 RNA-regulated molecular tweezers for sensitive fluorescent detection of microRNA from cancer cellsWe describe here the construction of the DNA self-assembled molecular tweezers and the application of the tweezers for the monitoring of microRNA(miR-141) from human prostate cancer cells. The self-assembly formation of the DNA tweezers and the regulation of the tweezers upon alternative addition of the fuel miR-141 and the anti-fuel strands are characterized by native polyacrylamide gel electrophoresis. The addition of mi R-141 to the DNA tweezers turns “off” the tweezers, while subsequent introduction of the anti-fuel strands switches the tweezers back to the “on” state, which verifies the regulatory ability of the tweezers. The miR-141-regulated DNA tweezers are concentration dependent and can be employed for sensitive detection of miR-141 down to 0.6 pM. The DNA tweezers also show high selectivity toward the fuel strand and can be used to monitor miR-141 expression in cancer cells, which provides new opportunities for the application of the dynamic DNA devices in clinical diagnostics. Part 2 RNA-induced cascaded and catalytic self-assembly of DNA nanostructures for enzym-free and sensitivefluorescent detection of microRNA from cancer cellsIn this work, we describe a facile strategy to utilize microRNA-triggered self-assembly formation of DNA nanowheel for ultrasensitive detection of microRNA(miR-141) from human prostate cancer cells. In the presence of target microRNA an autonomous without external intervention self-assembly DNA nanowheel can formation in a cascade simultaneous T-junction cohesion and hybridization strand reaction(HCR) manner. The triggered self-assembly behavior was monitered by agarose gel electrophoresis, and the effect of assemby time and temperature was optimized as well. Besides, the miR-141 is reused by toehold-mediated strand displacement to initiate self-assembly again to formation numerous fluorescent nanowhee leading to highly sensitive monitering of miR-141 down to 261 cancer cells. Our results may provide a versatile route for developed sophisticated DNA nanostructure with functional signal amplification to detection of wide-ranging of biomarkers. Part 3Terminal protection of small molecule-linked ssDNA for label-free and highly sensitive colorimetric detection of folate receptor biomarkersSmall molecule/protein interactions have a key role in drug discovery, clinic diagnosis and protein-metabolite interactions in biology. By using specific interaction between folic acid(FA) and folate receptor(FR) as a model, the development of a simple, label-free and sensitive colorimetric approach for the detection of the FR biomarker is described. The sensing approach relies on the coupling of the FR-induced terminal protection of FA-linked ssDNA strategy with significant signal amplification by self-assembled DNAzyme polymers. The FR binds to the FA-ssDNA and protects the FR/FA-ssDNA from digesting by exonclease I. The terminal protected ssDNA further triggers autonomous self-assembly of two G-quadruplex sequence-containing hairpin DNAs into DNAzyme polymers, which result in intensified color change of the probe solution for label-free and highly sensitive colorimetric detection of FR. The terminal protection mechanism and the self-assembly formation of the DNAzyme polymers are characterized by using polyacrylamide gel electrophoresis, and the sensing parameters are optimized as well. Under optimal experimental conditions, the detection limit of 0.35 pM for FR can be obtained by using a UV-Vis spectrophotometer and the presence of as low as 5 pM of FR can be directly visualized by naked eye. The developed method is also selective and can be applied to detect FR in serum samples, which makes this approach a sensitive platform for sensing different types of small molecule/protein interactions Part 4 Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymesBased on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules(ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar(0.33 nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.