Single-Molecule Detection Of Telomerase And M~6A Methyltransferase METTL3/14

Cancer is one of the public health problems that seriously endanger the physical and mental health of people all over the world.Researches demonstrate that the recovery rate of early cancer treatment is significantly higher than that of advanced cancer.Therefore,early detection and diagnosis is the key to prevent and control cancer.Tumor markers are critical indicators for early cancer diagnosis.Therefore,the sensitive detection of tumor markers plays an important role in early cancer diagnosis and anti-cancer drug development.Single-molecule detection technology is a rapidly emerging field in analytical chemistry,and the single-molecule counting is a part of single-molecule detection technology.By counting a single molecule,the ultrasensitive detection can be achieved.By using this technology,we can achieve simple,fast,low background,and highly sensitive detection of biomarkers.On this basis,the tumor markers（e.g.,telomerase and m⁶A methyltransferase METTL3/14）were detected by integrating the fluorophore-coded biosensor with single-molecule detection technology.The main contents are as follows:1.By taking advantage of in vivo repair mechanisms and single-molecule detection,we demonstrate the enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells.The presence of telomerase enables repetitive addition of（TTAGGG）n to the 3′-OH end of telomerase substrate（TS）primer to generate long telomeric products.Telomeric product then hybridizes with the apurinic（AP）probe to induce the cleavage of AP site in ds DNA by APE1,yielding a ss DNA with a biotin and an OH group at 5′and 3′ends and simultaneously releasing the telomeric product which in turn hybridizes with new AP probes to yield numerous ss DNAs.By adding streptavidin-coated MBs,the ss DNAs are captured and subsequently functioned as primers to initiate template-free Td T-catalyzed random incorporation of d ATPs and Cy5-d ATPs into 3′-OH ends,producing long poly-A chains with multiple Cy5molecules encoding and finally forming Cy5-ss DNA-MBs complexes.After magnetic separation,Cy5-ss DNA-MBs are digested by Exo I,releasing large numbers of Cy5 molecules which can be measured by single-molecule detection.This strategy achieves sensitive detection of telomerase in single cells,as well as specific detection of telomerase.Moreover,it can be employed to screen inhibitors and discriminate different cancer cell lines from normal cell lines,holding enormous potential in clinical diagnosis and therapeutics.2.We constructed a single quantum dot（QD）-based fluorescence resonance energy transfer（FRET）biosensor by using RNA methylation-driven DNA polymerase-mediated extension for accurate,stable,m⁶A-free recognition protein,and ultrasensitive detection of METTL3/14complex activity.In this biosensor,we designed a DNA embedded with 5 RNA nucleotides（5’-r Gr Gr Ar Cr A-3’）as the specific substrate for METTL3/14 complex.When METTL3/14 complex is present,it can catalyze the methylation of DNA substrate,preventing the cleavage by Maz F endonuclease.The methylated DNA substrate can hybridize with biotinylated linear template to initiate the polymerization-extension reaction with the assistant of DNA polymerase,forming a double-stranded DNA（ds DNA）with biotin-multilayer Cy5.Subsequently,the resultant ds DNA with multiple Cy5 molecules can self-assemble to the surface of 605QD to form 605QD-Cy5nanostructures,resulting in effective 605QD-Cy5 FRET.This biosensor can sensitive detect METTL3/14 complex with a detection limit of 3.1×10^-17 M,and can further detect METTL3/14complex in a single cell.Moreover,this biosensor can be applied for the measurement of METTL3/14 complex kinetic parameters and the screening of METTL3/14 complex inhibitors.More importantly,this biosensor can differentiate the METTL3/14 complex expression in breast cancer patient tissues and healthy person tissues,providing a powerful tool for cancer pathogenesis research,diagnosis,classification,prognosis,and anti-cancer drug development.