Nucleic Acid Probe Based Strategies For Ultrasensitive Signal Amplification And Efficient Cellular Delivery

In the past decades,nucleic acid probe has been developed rapidly as an important tool in the field of biological analysis.Nucleic acid probe with many advantages,such as good stability,sequence diversity,easy for synthesis and modification,has been widely used for the detection of nucleic acid,small molecules,metal ions,proteins,enzymes,transcription factors and even cells.Recently,with the development of nucleic acid amplification methods and nanomaterial technology,reaschers have spent efforts on these new thchnologys,which could improbe the capacity of nucleic acid probe,such as stability,sensitivity,and response rates.Morever,combing these new technologys with nucleic acid probe promote the application of nucleic acid probe in complex environment,for example,live cells or in vivo.By combining the nucleic acid probe with signal amplification strategy or nanomaterials thchnology,this thesis developed a series of novel biosening platforms for single nucleotide polymorphism(SNP)detection,simultaneous multicolor mRNA imaging,endogenous metal ions and tolemerase activity detection,with good selectivity and sensitivity.The mian contents are described as follows:1.Single-base Mismatch Discrimination by T7 Exonuclease with Target Cyclic Amplification DetectionSingle nucleotide polymorphism(SNP)is the most common genetic variation,and SNP detection is important for early diagnosis,clinical prognostics,and prevention.Rapid and accurate screening of SNP among individuals is still an unmet challenge in current society.In chapter 2,We first found that T7 exonuclease can efficiently distinguish the perfectly matched base pairs from the single-base mismatched pairs at the 5’ terminal of the double-strand DNA(dsDNA).we present a graphene oxide(GO)based approach using T7 exonuclease to discriminate single-base mutation for the first time.The detection was based on the hybridization between a single-strand DNA(ssDNA)target and an allele-specific DNA probe that labeled with a FAM fluorophore.For the perfectly matched dsDNA,T7 exonuclease activity shears the FAM-labeled probe generating a process of target recycling,resulting the fluorescence cannot be quenched by the GO nanosheets,however,no such phenomenon happened for the single-base mismatch dsDNA.We have detected as low as 4 pM of an SNP analyte which is related with Beta-Thalassemia disease.The results demonstrate that this system can precisely,sensitively,and low-costly discriminate the SNP.2.Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme-Catalytic Hairpin Assembly ProbeMetal ions play critical roles in numerous biological processes.1 Metal ions both in excess and deficient can be detrimental to their normal functions.To gain a better understanding of the functions of these metal ions in biology,it is important to detect and monitor their concentrations inside cells.DNAzymes have been shown as a promising platform for metal ions detection and a few DNAzyme-based sensors have recently been reported to detect metal ions in living cells.However,these methods can only detect metal ions after an influx of metal ions to increase their concentrations inside cells.To address the above major issue,in chapter 3,we report the design of a catalytic hairpin assembly(CHA)reaction to amplify the signal from photocaged Na+-specific DNAzyme cleavage to detect endogenous Na+ inside cells.Upon light activation and in the presence of Na+,NaA43 DNAzymes cleave the substrate strands and release initiator DNA that trigger the followed CHA amplification reaction.This strategy has allowed detection of endogenous Na+ inside cells,which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population.Since most DNAzymes that reported for metalions detection often share a similar secondary structure and reaction mechanism,the method demonstrated here potentially can be applied as a general strategy to improve the sensitivity of any DNAzyme-based sensor for detection of metal ions at low concentrations.As a result,the application of DNAzymes can be expanded for imaging many metal ions in biology,which will provide deeper understanding of the role of metal ions in biological systems.3.Electrostatic Fluoresent Blocked Conjugated Nanoparticle-DNA Nanoassemb-ly for Intracellular Multicolor mRNA ImagingMessenger RNA(mRNA)is important for many biological processes,such as induced cancer,where tiny minority of cells in a tumor mass can lead to major health troubles.Many mRNAs are tumor-related and can be used as specific biomarkers assessing the migration of tumor cells regionally or in the bloodstream.So it is significant to identify the changes of the level of tumor-related mRNA expression in living cells.In chapter 4,we developed an electrostatic fluoresence blocked con-jugated nanoparticle-DNA nanoassembly for intracellular multicolor mRNA imaging.By simultaneous imaging the tumor-related mRNA,it allowed avoiding the false positive signals.Meanwhile,with the dual-fluorescence from the nanoparticle and DNA probe,it allowed directly assessing the transfection process and avoiding the false negative signals.This novel sensing strategy provides a promising tool for biomarkers discovery and diagnosis.4.Electrostatic Nucleic Acid Nanoassembly for Direct Visualization of Telomerase Activity in Living CellsTelomerase is a ribonucleoprotein enzyme that catalyzes the addition of the telomeric repeats(TTAGGG)n onto the 3’-end of the human chromosomes by using reverse transcription and its intrinsic RNA as a template.Telomerase function is fundamental to the proliferation and differentiation of cells.Telomerase is known to related with various diseases such as cancer and stem cell disorders.The renders this enzyme an important therapeutic target and a valuable marker of malignancy and tumor progression.In chapter 5,we developed an electrostatic nucleic acid nanoassembly for direct visualization of telomerase activity in living cells.The DNA nanoassembly consists of a gold nanoparticle,a cationic peptide interlayer and the fluorophore-tagged DNA probes.The cationic peptide was modified on the surface of gold nanoparticle through Au-S bond.At the presence of telomerase,the DNA probe process a structure switch,resulting the florescence recovery.This novel electrostatic nuleic acid nanoassembly involved an endocytosis-independent cellular delivery mechanism with high efficiency,overcoming endosomal entrapment.This probe was demonstrated a good tool for tomelerse activity detection in living cells,providing a potential platform for biomarker detection and in vivo clinical diagnosis.