Bioanalysis And Biosensors Based On Functional Nucleic Acids

DNA is widely known as the carrier of genetic information in all known living organisms and many viruses.Besides that,it is capable of binding to specific targets and catalyzing specific biochemical reactions.The identification of the functional nucleic acids has attracted substantial research interests owing to their unique structures,catalytic abilities and mechanisms.They have shown great potential in analyzing a variety of targets.In this thesis,we focused on the basic research of functional nucleic acids,and their possible applications in biosensing.A series of novel biosensing strategies based on rolling circle amplification and strand displacement were developed for the detection of proteins,small molecule and nucleic acid,respectively.These results showed that the proposed technologies based on functional nucleic acids have great potential in providing platforms for applications in the fields of biosensing,biochemistry,clinical diagnostics and environmental sample analysis.The main sections of this dissertation are as follows:Based on the excellent stability of aptamer and its interaction with specific targets,a novel and versatile colorimetric platform for ultrasensitive detection of proteins has been developed.This method utilizes a circularized DNA which contains an aptamer domain as a regulatory unit to control Rolling circle amplification(RCA)which can generate a long single-stranded DNA(ss DNA)molecules with tandem repeats.When mixing the tandem repeats –PNA with the cyanine dye Di SC2(5),it can produce a colorimetric readout signal.This method can be adapted to the detection of proteins with high sensitivity and specificity.By utilizing the capability of arresting of RCA by aptamer-target interactions,we combined catalytic DNA(DNAzyme)and inhibitory RCA product and developed a widely applicable platform for the detection of different targets.By using an aptamer in conjunction with a circularized DNA template to function as a regulatory unit,RCA can be controlled.The combination of catalytic DNA and inhibitory RCA product to control fluorescence activity allows for a sensitive method of detection.A highly sensitive universal sensing platform for bothproteins and small molecules by combining aptamer-based RCA with an RNA-cleaving deoxyribozyme has been successfully developed.The self-phosphorylating deoxyribozymes identified by invitro selection can catalyze their own phosphorylation by utilizingphosphate donor guanosine-5’-triphosphate(GTP)which plays a criticalrole in a majority of cellular processes.On the basis of the unique properties of self-phosphorylating deoxyribozymes,we report a novel GTP sensor coupled with λ exonuclease cleavage reaction and nicking enzyme assisted fluorescence signal amplification process.Sensitive and selective detection of GTP was successfully realized.The method not only provides a platform for detecting GTP but also shows great potential in analyzing a variety of targets by combining deoxyribozymes with signal amplification strategy.In order to further utilize the unique properties and advantages of DNA for constructing sensing system,a novel miRNA-responsive biosensor was developed on the basis of strand displacement and RCA.micro RNAs(mi RNAs)are small RNA sequences that mediate post-transcriptional regulation of specific gene expression.As many members within the same mi RNA family share similar sequences and structures,it is still a challenge to distinguish between related miRNAs.We designed a mi RNA-responsive DNA walking biosensor based on mi RNA-triggered strand displacement cascades.This biosensor exhibits excellent analytical performance toward the sensing of let-7a with great specificity for resolving one nucleotide variation.