Development Of Fluorescent Biosensors For The Detection Of Nucleic Acid Modifying Enzymes

The enzymes that affect change in the nucleic acid chemical constitution or topology are generally referred to as nucleic acid modifying enzymes.Nucleic acid modifying enzymes include DNA modifying enzymes and RNA modifying enzymes,which play important roles in epigenetic regulation,cell cycle and heat shock stress response in eukaryotes.Aberrant expression of nucleic acid modifying enzymes is associated with a variety of cancers,such as breast cancer,ovarian cancer,lung cancer,colon cancer and urological cancer.Therefore,it can be acted as a biomarker for early cancer diagnosis.The development of efficient methods to detect nucleic acid modifying enzymes has far-reaching implications for biomedical research,clinical diagnosis and drug discovery.The conventional methods for nucleic acid modifying enzymes activity assay include radioactive labeling,enzyme linked immunosorbent assay,high performance liquid chromatography and western blot.However,they suffer from radioactive contamination,poor sensitivity and time-consuming procedures,which hardly meet the demand for sensitive detection of trace targets.Therefore,it is necessary to develop simple and sensitive methods for the detection of nucleic acid modifying enzymes.Fluorescent biosensors can convert the signal of biometric molecule-target interaction into a measurable fluorescent signal by specific in/ex vivo fluorescent labeling,which has the advantages of low sample consumption,high sensitivity and good specificity.Herein,we construct fluorescent biosensors to achieve sensitive detection of nucleic acid modifying enzymes.This thesis includes the following contents:（1）We construct a novel spinach aptamer RNA-based label-free fluorescent biosensor for ultrasensitive detection of uracil DNA glycosylase（UDG）.This biosensor only involved a single hairpin-structured probe,which include a double-stranded T7 promoter and a single-stranded spinach RNA template,with the upper and lower strands of the T7 promoter being connected by a T4（5’-TTTT-3’）linker.The T7 promoter is engineered through substituting deoxyribouracil for the thymine base near the junction between the recognition region and the initiation region in the upper T7 promoter strand.T7 RNA polymerase can bind with the T7 promoter of the probe and catalyzes the efficient transcription reaction through the incorporation of NTPs to synthesize abundant spinach RNAs.The resultant spinach aptamer RNA can bind to DFHBI to produce a distinct fluorescent signal.In contrast,when UDG is present,it can excise dU from the dU:A base pair,generating an apurinic/apyrimidinic（AP）site.The AP site is subsequently cleaved by AP site-specific endonuclease IV,leading to the destruction of both the recognition region and the initiation region of the promoter.Due to the lack of an intact promoter,the transcription reaction completely prohibited.Therefore,neither spinach aptamers nor fluorescent signals are produced.In this method,only a single DNA probe is required for both UDG signal recognition and amplification,greatly simplifying the reaction scheme without the involvement of multiple probes for signal amplification.And the RNA transcription reaction is extremely T7 promoter specific,effectively eliminating non-specific amplification and improving detection efficiency.Notably,the introduction of spinach aptamer RNA is a label-free way of signal output,eliminating expensive and complex fluorescent labeling.The constructed biosensor has excellent specificity and high sensitivity with a LOD of 6.3×10^-6 U mL^-1.In addition,this biosensor has been successfully applied for kinetic analysis,inhibitor screening and detection of endogenous UDG activity in different human cell lines,providing a potential application value for biomedical research and clinical diagnosis.（2）We establish a single-molecule fluorescent biosensor for ultrasensitive detection of RNA methyltransferase METTL3/METTL14 complex（METTL3/METTL14）activity based on a 3D DNA walker.In this biosensor,we rationally designed a fluorescent probe with 5’-terminus modified sulfhydryl group and 3’-terminus modified Cy5,which self-assembled onto the surface of gold nanoparticles via Au-S bonds to form super-quenched AuNPs@DNA nanostructures.The presence of METTL3/METTL14 can transfer the methyl group from S-adenosylmethionine to5’-G-G-A-C-A-3’of N⁶-adenine,to produce the methylated 5’-G-G-mA-C-A-3’which prevented cleavage by Maz F.The methylated RNA probe hybridizes with DNA fluorescent probes of AuNPs@DNA nanostructures and subsequently serves as the walker RNA to induce cyclic DSN-assisted cleavage of DNA fluorescent probes,liberating abundant Cy5 fromAuNPs@DNA nanostructures.Due to the high precision of Maz F-mediated digestion of the5’-G-G-A-C-A-3’sequence,high specificity of DSN-assisted DNA fluorescent probes cleavage,high efficiency of 3D DNA walkers and ultrahigh signal-to-noise ratio of single-molecule imaging,this biosensor exhibits a detection limit of 9.8×10^-6 nM for METTL3/METTL14.In addition,this biosensor can be used for METTL3/METTL14 inhibitor screening and kinetic parameter measurement.Importantly,it can quantify METTL3/METTL14 activity at single-cell level and discriminate cancer from normal tissues,which has potential applications in clinical diagnosis and drug development.