Development And Application Of Functional Nucleic Acids Based On Intramolecular Split G-quadruplex

Functional nucleic acids(FNAs)are nucleic acid molecules that have special structure or functions,such as G-quadruplex,i-motif,DNase and RNase,etc.The G-quadruplex as one kind of classical FNAs has been widely utilized in the field of analysis,because of its special structure and properties of ligand-specific fluorescence enhancement or peroxidase-like activity of the G-quadruplex-hemin complex(G4DNAzyme).Intermolecular split G-quadruplex(Inter-SG)is an important G-quadruplex-based analytical strategy.The Inter-SG-based binary molecular probes possess prominent recognition ability toward single-base mismatches,but the operation procedures are cumbersome and the repeatability is poor.Intramolecular split G-quadruplex(Intra-SG)was proposed on the basis of Inter-SG and is one new G-quadruplex-based analytical strategy.Unimolecular Intra-SG simplifies the design of molecular probes and reveals obvious advantages,e.g.,ease of design,high universality,and convenience of application.However,the present research on the interaction between Intra-SG structures and ligands has not been implemented sufficiently,which is not conducive to the application of Intra-SG to design of DNA molecular probes.Therefore,in this thesis,an Intra-SG was centered on,its interaction with ligands and property changes were systematically investigated.A series of phenomena and rules on the topological effect were disclosed,and novel DNA probes were obtained,on the basis of hybridization-induced the fluorescence light-up and catalytic regulation.Moreover,an ultrasensitive method for gene detection was constructed via coupling Intra-SG with DNA amplification techniques.The main research contents are listed as follows.1.Topological effect of an intramolecular split G-quadruplex on thioflavin T binding and fluorescence light-upTopological effect on binding interaction between G-quadruplex and Thioflavin T(ThT)ligand was systematically investigated on a platform of Intra-SG.Distinct fluorescence changes from ThT were presented in the presence of distinct split modes of Intra-SG structures and an intriguing phenomenon of target-induced fluorescent light-up occurred for split mode 2:10,5:7 and 8:4.It was validated that hybridization between Intra-SG spacer and target did not unfold the G-quadruplex,but facilitated the ThT binding.Moreover,it was demonstrated that 3′guanine-rich fragment of Intra-SG was much susceptible to topology variation produced by bound target strand.It is believed that sensitive detection of significant targets can be realized with the new functional nucleic acids based on ThT/Intra-SG,the results and disclosed rules will inspire researchers to develop much new DNA-based signal transducer in the future.2.Ultrasensitive gene detection by coupling an intramolecular split G-quadruplex with cascaded nucleic acid amplification circuitsBased on the aforementioned functional nucleic acid,Intra-SG(8:4),we explored a new ThT/Intra-SG transducer with high fluorescence response,demonstrated that the sensitivity was closely correlated with the sequence of the DNA spacer,and further validated that the susceptibility of 3′guanine-rich fragment of Intra-SG and the existence of a critical recognition sequence.In order to overcome the limitations,e.g.,low sensitivity and inhibition of protruding DNA,an ultrasensitive and selective method for detecting a gene segment from Hepatitis B virus(HBV)was constructed,through coupling hemin/Intra-SG(8:4)with two-fold amplification reactions including asymmetric polymerase chain reaction(A-PCR)and rolling circle amplification(RCA).The linear responsive range of the method was from 1 to 10~3copies.The data indicate the successful fabrication of the ThT/Intra-SG-based DNA probe,and predict its practical applications in the future.3.Tpological effect of an intramolecular split G-quadruplex on hemin binding and catalytic changesTopological effect on the catalytic properties of complexes between Intra-SG and hemin ligand was investigated with ABTS oxidation by hydrogen peroxide.Derived from a classic unimolecular G-quadruplex(PW17),eleven Intra-SG sequences of different split modes were designed.All these sequences folded into parallel G-quadruplex structures and exhibited apparent peroxidase-mimicking catalytic properties.Distinct changes of catalytic activity of hemin/Intra-SG were observed upon addition of a complementary strand of DNA spacer,and catalytic enhancement occurred for several Intra-SG(especially 5:7).It was verified that the hybridization triggered small topological change,and influenced the hemin binding and catalytic reaction.Analogous to ThT/Intra-SG,a critical recognition sequence also existed for hemin/Intra-SG.Subsequently,new DNA probes were designed for two disease-related micro RNA(M21 and Let-7a),based on the target-induced catalytic regulation of hemin/Intra-SG.