New Signal Transduction And Amplification Strategies Of Nucleic Acid Probes

In recent years, nucleic acid probe (NAP) have become a class of significant bioanalysis tools for its wide application and huge development potential. As a biosensor, NAPs are mainly built by nucleic acid sequences which can recognize targets by base pairing and other non-covalent interactions. Upon target binding, measurable signals, such as optical, electrical or magnetic signal can be output. However, direct conversion of target recognition events into signal readouts merely depends on one single step, which remains one of the bottlenecks for further improving the sensitivity of NAP sensors. In addition, intrinsic limitations, such as re-synthesis of several different capture probes, which are generally labeled with fluorescent or electrochemical signal elements will make the application costly and tedious.Therefore, generalizability remains a problem since the capture probe is sequence-specific and, hence, limited by the capture probe-binding target against which it can be deployed. To address these issues, triplex molecular switch and DNA hybridization chain reaction, combing with nucleases and host-guest interaction, are employed to construct a series of new general nucleic acid sensing platform in this thesis. Various analytes, such as metal ions, DNA, thrombin, and even cancer cells can be achieved sensitive detection. As further application, self-assembled multifunctional spherical nucleic acids (SNAs) were constructed to realize high drug loading and high specificity by the incorporation of an aptamer for cancer therapy. The main works are listed as follows:1) A hairpin nucleic acid molecular switch with inherent signal-transduction mechanism was designed, and KF-polymerase with properties of3’-end to5’-end polymerization and strand-displacement property was also employed for amplified detection of single nucleotide polymorphisms. The hairpin nucleic acid molecular switch itself acts as both template and primer of polymerization reaction upon the target addition. The target DNA acts as a kind of special "catalyst" to trigger a conformational change of the otherwise inactive structure into a polymerization responsible active structure. In the process of the polymerization reaction, the target DNA was displaced and then hybridized to trigger another probe. Thus, this strategy allows hybridization, polymerization reaction and displacement to occur cycle after cycle, producing, at the same time, a sufficient amplified signal to indicate the presence of trace target. 2) We proposed a way to restrict the labeled-pyrene in a hydrophobic cavity of cyclodextrin based on host-guest interaction. This bounding, which acts like extra base pairs to form Watson-Crick duplex, achieves variation of level of space proximity of the two labels and thus the degree of conformational constraint. This approach is simple in design, avoiding any variation of the stem’s length and sequences. Furthermore, the strategy is generalizable which is suited for not only the stem-containing probe but also the linear probe with comparable sensitivity and selectivity to conventional structured DNA probes.3) We proposed a series of nucleic acid-based fluorescent sensing platform combing with triple-helix molecular switch.i) We reported a new type of aptamer-based sensing platform which is based on triple-helix molecular switch (THMS). The THMS consists of a central, target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide serving as a signal transduction probe (STP). The STP is doubly labeled with pyrene at5’-and3’-end, respectively, and initially designed as a hairpin-shaped structure, thus, bringing the two pyrenes into spacer proximity. Bindings of two arm segments of the aptamer with the loop sequence of STP enforce the STP to form an "open" configuration. Formation of aptamer/target complex releases the STP, leading to new signal readout. The universality of the approach is achieved by virtue of altering the aptamer sequence without change of the triple-helix structure.ii) We present a time-resolved emission spectra-based approach for Hg2+detection involving the long lifetime of pyrene excimer emission, which can be easily distinguished from short-lived fluorophores by time resolved fluorescence spectra (TRES). In our design, a thymine (T)-rich mercury-specific oligonucleotide (MSO) was selected as the captured probe. A bis-pyrene-labeled oligonucleotide was used as a signal transduction probe (STP). To further effectively eliminate pyrene excimer fluorescence quenching by Hg2+, magnetic nanoparticle conjugated-MSO was firstly hybridized with STP to form a hairpin-shaped THMS through Watson-Crick and Hoogsteen base pairing, the central sequence is exposured as a loop. Binding of Hg2+with the MSO will release the STP, leading to pyrene excimer emission as a result of the conformation rearrangement of the STP. Combining TRES assays, short-lived background fluorescence from sample can be eliminated efficiently, thus this approach could be used to directly and sensitively detect trace Hg2+in biological samples. 4) We proposed a series of nucleic acid-based surface enhanced Raman scattering (SERS) sensing platform combing with triple-helix molecular switch.i) We report, for the first time, a reversible and regenerable SERS hot spot employing DNA-and Raman dye-functionalized AgNPs on an Au film surface. On the basis of a configuration switch between an "open" and a "duplex-stem" cyclic DNA structures, the hot spot effectively switches from an "off to "on" signal state following the action of a biological recognition event. Two triggers of nucleic acid sequences associated with the LTR and ATP were examined to induce hot spot generation, which, by virtue of the ability to alter the MRP sequence, is applicable to other nucleic acids, proteins, and small molecules for biomedical applications,ii) We described a universal aptamer-based SERS biodetection approach that uses a single-stranded DNA as a universal trigger (UT) to induce SERS-active hot-spot formation, allowing, in turn, detection of a broad range of targets. More specifically, interaction between the aptamer probe and its target perturbs a triple-helix aptamer/UT structure in a manner that activates a hybridization chain reaction (HCR) among three short DNA building blocks that self-assemble into a long DNA polymer. The SERS-active hot-spots are formed by conjugating4-aminobenzenethiol (4-ABT)-encoded gold nanoparticles with the DNA polymer through specific Au-S bond.5) As further application, we engineered a nanoparticle-conjugated initiator that triggers a cascade of hybridization reaction resulting in the formation of a long DNA polymer as the nanoparticle shell. By employing different DNA fragments, self-assembled multifunctional spherical nucleic acids (SNAs) can be constructed. Therefore, using one capped ligand, these SNAs can combine imaging fluorescent tags, target recognition element, and targeted delivery molecules together. Since these SNAs possess high drug loading capacity and high specificity by the incorporation of an aptamer, our approach might find potential applications in new drug development, existing drug improvement, and drug delivery for cancer therapy.These strategies are expected to supply valuable concepts to develop key techniques and fundamental research in biosensor with the advantages of high specificity and high sensitivity, thus achieve multiple analytes detection.