Investigation Of New Nanoassembly And Biosensors Based On Functional Nucleic Acids

Functional nucleic acids include two types of nucleic acid molecules, one type ofthem can bind to a specific target molecule like antibodies (called aptamers), and theother have catalytic reactions like protein enzymes (called DNAzymes, ordeoxyribozymes). In the traditional understanding, the nucleic acids are only thestorage and transfer carrier of genetic information. However, the discovery offunctional nucleic acids has led to a breakthrough, and provided an interestingalternative to biosensing system.Recently, nanomaterials have been regarded as the research focus due to theirexcellent optical and electrochemical properties. DNA is a kind of nanomaterial,which can bring an unprecedented role for life science, material science,environmental science and other fields. Therefore, it has received more and moreattention. For example, DNA molecules have been utilized both as powerful syntheticbuilding blocks to create nanoscale architectures and as versatile programmabletemplates for assembly of nanomaterials. However, these conventional approacheshave some intrinsic drawbacks.Complicated design resulting from the myriad ofdifferent DNA strands needed to assemble relatively large and sophisticatednanostructures. So the large amount of DNA needed for bulky preparation. It willdissociate extremely low concentrations, such as that in an in vivo circulation system,resulting in loss of nanostructure integrity. How to synthesize the new DNAnanomaterials that are easy to synthesize, highly water-soluble, biocompatible andhighly-stable has been an unsolved problem for researchers.Nucleic acids are stable and adaptable and they can be easily produced using acommercial DNA synthesizer. Given the tremendous progress made in both DNAnanotechnology and in the study of functional DNA, it is natural to combine thesetwo exciting fields to create a new interdisciplinary field that synthesis new DNAmaterials produce novel, simple and low-cost biosensors and delivery drug totargeted cells.The details are summarized as follows:(1) In chapter2, A DNAzyme-based ELISA, termed DLISA, was developed asa novel protein enzyme-free, triple-amplified platform, combining a catalytic andmolecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs proteinenzymes as biocatalysts to afford amplified signals, suffers from poor stabilitycaused by the irreversible denaturation of these enzymes under harsh conditions,such as heat and acidity. Compared with proteins, nucleic acids are more stable andadaptable and they can be easily produced using a commercial DNA synthesizer.Moreover, the catalytic and cleavage activities of DNAzyme can be achieved insolution; thus, no enzyme immobilization is needed for detection. Taken together,these attributes suggest that a DNAzyme-based ELISA detection approach will bemore robust than current ELISA assays. Importantly, the proposed triple-amplifiedDLISA immunoassay method shows ultrasensitive detection of such targets as humanIgG with a detection limit of2fg/mL, which is well within the range of manyimportant disease biomarkers. DLISA can also be used to construct a sensing arrayfor simultaneous multiplexed detection. With these merits, this high-throughput,stable, simple, sensitive and low-cost multiplex fluorescence immunoassay showspromising potential for applications in clinical diagnosis.(2) In chapter3, using double-stranded DNA-copper nanoparticles as signalreporters, a label-free biosensor for the sensitive detection of nuclease was developed.The double-stranded DNA could act as an efficient template for the formation ofcopper nanoparticles with high fluorescence, whereas the single-stranded DNAcannot support the formation of copper nanoparticles. This difference in fluorescentsignal generation can be used for the detection of nuclease activity. The enzyme S1nuclease was taken as the model analyte. S1nuclease is known to be asingle-strand-specific endonuclease. Upon the addition of the S1nuclease into thesensor, the DNA was cleaved into fragments, preventing the formation of the coppernanoparticles and resulting in low fluorescence. Under the optimized experimentalconditions, the sensor exhibited excellent performance (e.g., a detection limit of0.3U mL1).(3) In chapter4, aggregated cationic perylene diimide (PDI) derivative wasfound to efficiently quench the fluorescence emission of a variety of anionicoligonucleotide-labeled fluorophores that emit at wavelengths from the visible toNIR region. This broad-spectrum quencher was then adopted to develop a multicolorbiosensor via a label-free approach for multiplexed fluorescent detection of DNA..By combining aggregated perylene derivative broad-spectrum quenching with theExo III-assisted autocatalytic target recycling amplification strategy, we proposed asensitive and multiplexed analytical platform with post-addition approach for target DNA detection. The quencher did not interfere with the catalytic activity of nuclease,and the biosensor could be manipulated in either pre-addition or post-additionmanner with similar sensitivity. Moreover, the proposed sensing system allows forsimultaneous and multicolor analysis of several oligonucleotides in homogeneoussolution, demonstrating its potential application in the rapid screening of multiplebiotargets.(4) In chapter5, we report the nature-inspired noncanonical self-assembly ofmultifunctional hierarchical DNA nanoflowers (NFs) through liquid crystallizationand dense packaging of elongated DNA building blocks, and the diverse biomedicalapplications. The DNA building blocks were generated via Rolling Circle Replication(RCR). NF assembly does not rely on Watson-Crick base-pairing between DNAbuilding blocks, thereby circumventing the conventional complicated sequencedesign, and, combined with the size tenability of NFs (diameters: ca.200nm-4μmin our study), making this strategy widely applicable. NFs are exceptionally resistantto nuclease degradation and denaturation, as well as dissociation at extremely lowconcentration, which are mainly attributed to the intrinsic non-nicked elongated DNAbuilding blocks and the high density of DNA in NFs. By rational design, NFs can beincorporated with myriad functional moieties. The implementation of multifunctionalNFs was demonstrated for specific cancer cell recognition, bioimaging, and targetedanticancer drug delivery.(5) In chapter6, we presented a facile approach to making aptamer-conjugatedFRET-nanoflowers (NFs) via Rolling Cycle Replication for multiplexed cellularimaging and traceable targeted drug delivery. FAM, Cy3and ROX weresimultaneously incorporated into NFs via chemically modified deoxynucleotides,including FAM-dUTP, Cy3-dUTP and ROX-dUTP, during RCR reaction. The NFscan exhibit multifluorescence emissions by a single-wavelength excitation as a resultof the DNA matrix covalently incorporated with three kinds of dyes able to performFRET. Compared with the conventional DNA nanostructure assembly, NF assemblyis independent of template sequences, avoiding the otherwise complicated design ofDNA building blocks that were assembled into nanostructures via base-pairing. TheNFs were uniform and exhibited high fluorescence intensity and excellentphotostability. Combined with the ability of traceable targeted drug delivery, thesecolorful DNA NFs provide a novel system for potential applications of fluorescentnanoparticles in multiplex fluorescent imaging, effective screening of drugs andtherapeutic protocols for diseases.