Development Of Isothermal Amplification For Sensitive Detection Of 8-hydroxyguanine Glycosylase And Micro RNA

Nucleic acid amplification represents the most direct and effective way for target signal amplification.Isothermal amplification of nucleic acids proceeds at a constant temperature without the thermocycling required in polymerase chain reaction.Isothermal amplification provides rapid and high amplification efficiency,and a variety of amplification strategies have been introduced for highly sensitive biosensing.In this thesis,we use DNA glycosylase and microRNA as the model to develop two isothermal amplification-based methods for sensitive detection of targets.This thesis contains following contents:1.Simple mix-and-read assay with multiple cyclic enzymatic repairing amplification for rapid and sensitive detection of DNA glycosylase.We develop a simple mix-and-read assay for sensitive detection of DNA glycosylase based on multiple cyclic enzymatic repairing amplification.The human 8-oxoguanine DNA glycosylase（hOGG11）can initiate base excision repair of genomic 8-oxoguanine（8-oxo G）,and it can locate and remove damaged 8-oxo G through extrusion and excision.Sensitive detection of hOGG11 is critical for clinical diagnosis.In this work,hOGG11 can excise 8-oxo G base of the substrate to generate an apurinic/apyrimidinic（AP）site,and then the AP site can be cleaved by apurinic/apyrimidic endonuclease 1（APE1）,producing the substrate fragment with free 3’-OH terminus.Subsequently,the substrate fragment can initiate cyclic enzymatic repairing amplification,generating two triggers.The resultant two triggers can function as the primers to induce two cyclic enzymatic repairing amplification,respectively,producing more and more triggers.We experimental verify the occurrence of each cyclic enzymatic repairing amplification and UDG-mediated exponential amplification.The amplification products can be simply detected using SYBR Green II as the fluorescent dye.This mix-and-read assay is very simple and rapid（within 40 min）,without the requirement of any extra primers and modification/separation steps.This method can sensitively measure hOGG11 with a detection limit of 2.97×10^-8 U/μL,and it can be used for the screening of enzyme inhibitors and the measurement of hOGG11 in cancer cells at the single-cell level,providing a promising platform for clinical diagnosis and drug discovery.2.Sensitive detection of microRNA with isothermal amplification and a gold nanoparticle-based chemiluminescent biosensor.We develop a highly sensitive microRNA（miRNA）assay based on rolling circle amplification（RCA）and a gold nanoparticle（AuNPs）-based chemiluminescent biosensor.miRNA is a small class of non-coding RNA molecules found in eukaryotic cells.They are post-transcriptional regulators that bind to complementary sequences on the target mRNAs,resulting in translation inhibition,target gene degradation and gene silencing.Recent research reveals the aberrant expression of some tumor-specific miRNAs such as overexpression of miRNA-200b in several types of tumors.Therefore,there is an urgent need to develop a new sensitive method for miRNA detection.In the presence of miR-200b,the 3’-and 5’-terminal of the miR-200b-specific linear padlock probe can be ligated to form a circular template.Mi R-200b can function as a primer to initiate the RCA reaction in the presence of DNA polymerase,accompanied by the incorporation of abundant biotins in the extension product with the assistance of the biotin-labeled d UTP.The subsequent hybridization of extension product with the Au NP-modified capture probe and the addition of streptavidin modified alkaline phosphatase（STV-ALP）induce the assembly of large amounts of ALP onto the AuNPs via specific biotin-streptavidin binding.After separation,strong chemiluminescence signal of AMPPD can be detected.Due to the introduction of large amounts of biotins by miR-200b-induced RCA and highly specific chemiluminescence for ALP,this biosensor can sensitively detect miR-200b with a detection limit of 1.08×10^-15 M.This AuNPs-based chemiluminescent biosensor may become a useful analysis tool for the detection of other nucleic acids,holding great potential in the clinical diagnosis of genomic variation in various diseases.