| Since1953, the double-helix structure of DNA was proposed by James D. Watsonand Francis Crick, the research of life science has stepped into the molecular level, andwith that came the DNA hybridization technique. Nucleic acid probes play criticalroles in the development of this technique. Among different nucleic acid probes,oligonucleotides have attracted much attention, due to their intrinsic advantagesincluding short sequence, easy synthesis, high production yield, rapid response andgood selectivity. On the other hand, because of the one-to-one response ratio, thesensitivity is restricted to some extent. How to improve the sensing performance ofnucleic acid probes has been an unsolved problem for researchers.With the development of a novel in vitro selection method--systematic evolutionof ligands by exponential enrichment (SELEX), many functional nucleic acids havebeen screened, extending the application scope of traditional nucleic acids. Aptamersare functional oligonucleotides selected by SELEX on the basis of their specificaffinity to target cargos. These target cargos can be various small molecules, peptides,proteins, and even the whole cells. Upon specific binding with the target cargo s,aptamers can form a unique3D configuration. By using appropriate signal transductionmethods, several aptamer-based sensing systems have been developed. Whereas, theapplication of aptamer in the biosensing area is still in the infant stage.In addition to aptamers, DNAzymes are another functional oligonucleotidesselected by SELEX based on their ability to induce catalyti c reactions in the presenceof specific metal ions. Based on their specific recognition with target metal ion,several DNAzyme-based sensors have been developed for detecting metal ions,avoiding the complicated pretreatments of traditional methods. Wherer as, theapplication of DNAzymes in the biological systems is still limited.To solve the above-mentioned problems, we have developed several novelfunctional nucleic acid probes for detecting specific disease-related chemical speciesin different systems with high sensitivity and high selectivity. The details aresummarized as follows:(1) The existing isothermal polymerization-based signal amplification assays areusually accomplished via two strategies: rolling circle amplification (RCA) andcyclical strand-displacement polymerization. In essence, the two techniques are based on the cyclical nucleic acid strand-displacement polymerization (CNDP), limiting theapplication of isothermal polymerization in medical diagnosis and bioanalysis. InChapter2, cyclical common target molecule (non-nucleic acid strand)-displacementpolymerization (CCDP) is developed to amplify the fluorescence signal forbiomolecule assays, extending the isothermal polymerization to aptameric systemwithout any medium. Via combining an aptamer with common hairpin DNA probe, wedesigned a self-blocked fluorescent bifunctional oligonucleotide probe (signalingprobe) for the homogeneous parallel detection of two disease-relatted species,PDGF-BB and p53gene. On the basis of CNDP and CCDP signal amplification,highly-sensitive (e.g., detecting PDGF down to the concentration level of1.8×10-10M)and selective detection (no interference even in the presence of significantly higherconcentration (7~200times) of nonspecific proteins) were accomplished and the linearresponse range was considerably widened. Besides, the bifuncti onal signaling probeexhibits impressive simplicity, convenience and short detection time. Herein, thedesign of signaling probe was described, factors influencing fluor escence signal wereinvestigated, analytical properties were characterized in detail and the assayapplication in a complex medium was validated. The proposed biosensing scheme as aproof-of-concept is expected to promote the application of oligonucleotide probes inbasic research and medical diagnosis.(2) In Chapter3, we developed a multiple amplification-based electrochemicalsensor for the ultra-sensitive detection of nucleic acids, using a disease-relatedsequence of p53gene as the model target. A capture probe (CP) with hairpin structureis immobilized on the electrode surface via the thiol-gold bonding, whose stem isdesigned to contain a restriction site for EcoRI. In the absence of target DNA, theprobe keeps a closed conformation and forms a cleavable region. After treated withEcoRI, the target binding portion (loop) plus the biotin tag can be peeled off,suppressing the background current. In contrast, in the presence of target DNA, the CPis opened by the target hybridization, deforming the restriction site and forcing thebiotin tag away from the electrode. Based on the biotin-streptavidin complexation,gold nanoparticles (GNPs) modified with a large number of Fc-signaling probes arecaptured by the resulting interface, producing an amplified electrochemical signal dueto the GNPs-based enrichment of redox-active moieties. Furthermore, Fc tags can bedragged in close proximity to the electrode surface via hybridization between thesignaling probes and the CP residues after EcoRI treatment, facil itating interfacialelectron transfer and further enhancing the signal. With combination of these factors, the present system is demonstrated to achieve an ultrahigh sensitivity of zeptomolelevel and a wide dynamic response range of over seven orders of m agnitudes.(3) High-sensitivity and high-selectivity bioassay for single nucleotidepolymorphism (SNP) genotyping is of vital importance for the early diagnosis andeffective therapy of many diseases, especially cancer. In Chapter4, we developed anelectrochemical SNP genotyping sensor for the analysis of cancer-related genesequence. To improve the detection sensitivity, an effective signal amplificationstrategy was developed via combining gold nanoparticle (GNP)-based enrichmenteffect with the surface hybridization-based dragging strategy. With a large number offerrocene (Fc) probes enriched by GNP and then dragged in close proximity to theelectrode surface through DNA hybridization, a detection limit of femtomolar level (4fM,40zmol in10μL sample) can be achieved with a wide dynamic range (from10fMto1nM). By using the allele-specific nucleotide ligation, high selectivity fordifferentiating single point mutation was verified. Moreover, PCR amplicons from b othpatient samples and reference cell lines have been successfully tested, demonstratinggreat potential of this sensing scheme in medical diagnosis of genetic diseases(4) Cell membrane-anchored biochemical sensors that allow real-time monitoringof the interactions of cells with their microenvironment would be powerful tools forstudying the mechanisms underlying various biological processes, such as cellmetabolism and signaling. Despite the significance of these techniques, unfortunately,their development has lagged far behind due to the lack of a desirable membraneengineering method. In Chapter5, we proposed a simple, efficient, biocompatible anduniversal strategy for one-step self-construction of cell-surface sensors usingdiacyllipid-DNA conjugates as the building and sensing elements. The sensors exploitthe high membrane-insertion capacity of a diacyllipid tail and good sensingperformance of the DNA probes. Based on this strategy, we have engineered specificDNAzymes on the cell membrane for metal ion assay in the extracellular microspace.The immobilized DNAzyme showed excellent performance for reporting andsemiquantifying both exogenous and cell-extruded target metal ions in real time. Thismembrane-anchored sensor could also be used for multiple target detection by havingdifferent DNA probes inserted, providing potentially useful tools for versatileapplications in cell biology, biomedical research, drug discovery and tissueengineering.(5) The spatiotemporal dynamics of specifc mRNA molecules are difficult toimage and detect inside living cells, and this has been a signifcant challenge for the chemical and biomedical communities. To solve this problem, in Chapter6, we havedeveloped a targeted, self-delivered, and photocontrolled aptamer-based molecularbeacon (MB) for intracellular mRNA analysis. An internalizing aptamer connected viaa double-stranded DNA structure was used as a carrier probe (CP) for cell-specifcdelivery of the MB designed to signal target mRNA. A light activation strategy wasemployed by inserting two photolabile groups in the CP sequence, enabling controlover the MB’s intracellular function. After the probe was guided to the target cell viaspecifc binding of aptamer AS1411to nucleolin on the cell membrane, lightillumination released the MB for mRNA monitoring. Consequently, the MB is able toperform live-cell mRNA imaging with precise spatiotemporal control, while the CPacts as both a tracer for intracellular distribution of the MB before photoinitiation andan internal reference for mRNA ratiometric detection. |
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