Construction Of Nucleotide Signal Amplification-based Fluorescence Biosensors For Ultrasensitive Detection Of DNA-modifying Enzymes

Genomic DNA,constituted of Watson Crick-pairing heterocyclic bases,is the main carrier of genetic information whose accuracy and maintenance are fundamental prerequisites for all organisms.DNA-modifying enzymes are responsible for the cleavage,labeling,ligation,and extension of genomic DNA,and they participate in a variety of cellular processes including cell proliferation,differentiation,senescence,apoptosis.Malfunction of DNA-modifying enzymes may directly disturb the normal function of DNA replication,repairing and recombination,and eventually induce various human diseases including cancers.Therefore,DNA-modifying enzymes may function as the valuable biomarkers for disease diagnosis and potential targets for anticancer therapy and drug screening.The detection of DNA-modifying enzymes is crucial for fundamental biomedical research,clinic diagnosis,and anticancer-drug discovery.In this thesis,we developed two nucleotide signal amplification-based fluorescence biosensors for alkaline phosphatase（ALP）and DNA methyltransferase（DNA MTase）.The contents are as follows:（1）3’-Terminal Repair-Powered Dendritic Nanoassembly of Polyadenine（A）Molecular Beacons（MBs）for One-Step Quantification of ALP in Human Serum.ALP is an important hydrolase with crucial roles in biological processes,and the dysregulation of ALP may cause various human diseases.The conventional ALP assays usually involve cumbersome procedures with poor sensitivity.Herein,taking advantage of intrinsic superiorities of MBs and unique features of terminal deoxynucleotidyl transferase（Td T）,we demonstrate for the first time the 3’-terminal repair-powered dendritic nanoassembly of polyadenine MBs for one-step quantification of ALP in human serum.When ALP is present,it catalyzes 3’-terminal dephosphorylation of poly-A MBs to induce Td T-mediated template-free polymerization,generating long chains of polythymidine（T）sequences.The long poly-T chains can function as the anchoring templates to hybridize with many poly-A MBs,leading to the unfolding of loop structures and the dissociation of FAM/BHQ1 pairs（the 1st amplification stage）.Subsequently,all 3’-hydroxylated poly-A MBs can be extended with the assistance of Td T to generate the branched long poly-T chains,leading to the hybridization of more poly-A MBs and the dissociation of more FAM/BHQ1 pairs（the 2nd amplification stage）.Through multiple rounds of extension,assembly,and activation of poly-A MBs,dendritic DNA nanostructures are automatically formed,resulting in the dissociation of abundant fluorophores from the FAM/BHQ1 pairs to generate an exponentially amplified fluorescence signal（the nth amplification stage）.This strategy can evaluate kinetic parameters,screen inhibitors,estimate cellular inhibition effects,and measure ALP in human serums.（2）Methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases.DNA methylation is one of the most important epigenetic modifications and it is involved in regulating multiple cellular processes in both prokaryotes and eukaryotes.DNA MTases maintain methylation patterns in genomic DNA,and their aberrant activities may lead to various diseases including cancers.However,due to the diversity of catalytic substrates and the rareness of methylation-dependent restriction endonucleases（MDREs）,the accurate and sensitive detection of multiple DNA MTases still remains challenging.Herein,inspired by the intrinsic superiorities of light-up RNA aptamers and unique properties of T7-based transcription amplification,we demonstrate for the first time the methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases.In the presence of Cp G（M.Sss I）and adenine MTases（Dam）,the dumbbell probe 1（DP1）is methylated at the cytosine（C-5）and adenine（N-6）to form the catalytic substrates for Gla I and Dpn I,respectively,followed by simultaneous cleavage by Gla I and Dpn I to yield two cleavage products（CP1 and CP2）.CP1 and CP2 can hybridize with the 3’-end sequences of loops in DP2,and act as the primers to initiate bidirectional（strand displacement amplification,SDA）reactions,generating triggers 1 and 2.Triggers 1 and 2 can reversely hybridize with the5’-end sequences of stems in DP2,and function as the transcription templates to activate double transcription amplifications with the assistance of T7 RNA polymerase,producing two light-up RNA transcripts（i.e.,Mango and Spinach aptamers）.TO1-3PEG-biotin（TO1-biotin）and3,5-difluoro-4-hydroxybenzylidene imidazolidinone（DFHBI）can specifically bind to Mango and Spinach aptamers and generate enhanced dual-color fluorescence signals for simultaneous detection of M.Sss I and Dam MTases activities.This nanosensor exhibits good specificity and high sensitivity with a detection limit of 3.77×10^-3 U m L^-1 for M.Sss I MTase and 7.21×10^-5 U m L^-1 for Dam MTase.Furthermore,it can be applied to screen potential inhibitors,and quantify M.Sss I and Dam MTases activities from E.coli cells and human serum samples.Importantly,this nanosensor can be completed homogeneously in one tube within 2 h and detected in a label-free manner without any methylation-independent cleavage,sophisticated operation,and fluorescence tagging,promising a facile,novel and powerful platform for sensing multiple DNA MTases in biomedical studies and clinical therapeutics.