Novel Methods Of Optical Analysis Based On G-quadruplex Probe And Strand Displacement Amplification

Nucleic acids are important biological macromolecules essential for life, which arefound in abundance in all living things. As the development of synthesistechnology in vitro, especially with the discovery of the solid phase synthesis,nucleic acids have become important molecular tools and play important roles inbiochemical analysis. With the advantages of stable, biocompatible, simple design,easy to synthesize and flexible signal mechanism, nucleic acids have been widelyapplied in designing molecular probe, constructing molecular machines andbiosensors. Additionally, by combining with various tool enzymes, they can also beused in developing efficient signal amplification strategies. Nowadays, nucleic acidsbased molecular probe and signal amplification strategy have become importantelements of photochemical biosensor. This kind of biosensors has wide applicationprospects in chemistry, biomedicine, environmental monitoring, food industry,medicine and military affairs, attributing to its high sensitivity, easy operation, fastresponse, low cost and easy to realize high throughput analysis and miniaturization.Therefore, developing nucleic acid probe with multifunctions and efficient signalamplification strategy is vital for photochemical biosensor with excellent properties.We focused on these points and developed several photochemical biosensors in thispaper. The details are summarized as follows:(1) In chapter2, single fluorescein (FAM)-labeled G-rich signaling probewithout any quencher is developed as the model. This probe was designed to possessa continuous seven-guanine fragment (G7s) and a random tail. Its intermolecularparallel G-quadruplex (IGQ) structure is validated by recording fluorescenceemission, CD, UV, and gel electropherogram. Moreover, the developed probepossesses low self-quenching-based background fluorescence, and the restoration ofquenched fluorescence can be easily accomplished in a simple manner. Excitingly,more than90-fold fluorescence enhancement can be detected for an improvedIGQ-structure-based probe after hybridization to complementary strand, dramaticallyhigher than signal of traditional molecular beacon. The solid data imply thefascinating potential application of the concept of single fluorophore-labeled G-richprobe in biosensor development and G-quadruplex researches to acquire the furtherinformation on the function of vital nucleic acids in biological processes.(2) In chapter3, we further applied IGQ signaling probe to bind with an unmodified hairpin sequence and fabricated a novel quencher free molecular beacon(called intermolecular G-quadruplexb-based molecular beacon, IGQ-MB) for detectingof human P53gene. In this novel strategy, the molecular recognition element (hairpinsequence) and signal reporter has been successfully separated. Therefore, in practicalapplication, users just need to resynthesis the unmodified hairpin sequences accordingto the various requirements in optimization of MB or in detection of different targets,which obviously reduce the cost and improve the convenience. The novel strategy hasgreat potential in disease diagnosis, drug discovery and solid surface analysis.(3) There is a template/primer independent non-specific reaction existing in moststrand dispalsment amplification (SDA)-based detection systems, especially for theone with intercalating dye as the signal reporter, which seriously interference thebackground signal of the detection system. In Chapter4, we found that suchnon-specific reaction can be effectively inhibited by adding a special protein into thesystem. Furthermore, a novel SDA-based strategy, called nicking-induced reversingSDA model, is developed as a proof-of-concept. The model is mainly based on anintegrated template probe which is distinguished from tranditional multiprobe basedSDA system. The novel label-free SDA-based strategy is not only simple andinexpensive, but also suitable for detection of DNA and protein under the conditionof37°C.(4) In chapter5, we pointed out and demonstrated that there is a “signalmisreading” behavior in existing autonomous machines where the target recognitionprocess and signal transduction is separated from each other. We further developed aintegrated signal transduction-based autonomous aptameric machine, in which therecognition element and signal reporters are integrated into a DNA strand. This newmachine can execute the in situ amplification of target-induced signal. The authenticoperation behavior of autonomous DNA machine is discovered: the machine’s productsdirectly hybridize to the “track” rather than to the signaling probes. Along this line, themachine is employed to detect the cocaine in a more straightforward fashion, andimproved assay characteristics (for example, the dynamic response range is widenedby more than500-fold) are achieved. Our efforts not only clarify the concept describedin traditional autonomous DNA machines but also have made technologicaladvancements that are expected to be especially valuable in designing nucleicacid-based machines employed in basic research and medical diagnosis.(5) In chapter6, a powerful pendulum-type DNA (LPOD) nanoswitch, which canperform a reversible on/off molecular motion at an about9.4-nm scale, is developed as a proof-of-concept, and the sequence-specific recognition and sensitive quantificationof target olignucleotides are demonstrated utilizing this screening scheme. In contrastto the existing nanomachines (e.g. tweezers), the hybridization of the fuel strand withthe nanodevice opens the hairpin structure and straightens one arm, bringing thequencher into the close proximity of the fluorophore for FRET. Additionally, thepresent scheme has eliminated dimers as only one arm is kept in the single-strandedstate avoiding the hybridization of nanodevices with each other. Furthermore, ashybridization reaction occurring at a single “binding site” rather than the dual “bindingsites” eliminates the effect of spacer segments that can encountered for an integratedsignaling probe, the nanomachine seems to exhibit significant advantages.