Detection Of Telomerase As Well As Fat Mass And Obesity-Associated Protein By Single-Molecule Counting

Compared with traditional methods,single-molecule fluorescence detection technology has the advantages of high accuracy,low background and high sensitivity.Single-molecule fluorescence detection technology has been widely used for the detection of various biomarkers including DNA,RNA and proteins,holding great promise for applications in drug development and early diagnosis of diseases.In this paper,two fluorescent biosensors for sensitive detection of telomerase as well as fat mass and obesity-associated protein(FTO)were developed based on single-molecule counting.The research content is as follows:(1)We demonstrate the integration of telomerization-induced ligation-transcription circuit-driven cascade signal amplification with single-molecule detection for simple and sensitive measurement of telomerase in cancer cells.Telomerase is a ribonucleoprotein complex which is inactive in human normal somatic cells but is activated in a vast majority of cancer cells to maintain their infinite proliferation and immortal phenotype,making telomerase a promising diagnostic and therapeutic target.In this work,the presence of telomerase catalyzes the addition of tandem repeats(TTAGGG)_nto the 3’-OH of telomerase substrate(TS)primer to produce long-chain telomeric extension product(TEP).The generated TEP initially active the ligation-transcription circuit which enables the conversion of target events into numerous reporter RNAs.The released reporter RNAs then initiate the downstream DSN-assisted cyclic cleavage of signal probes which may serve as signal transducers and amplifiers to liberate large amounts of Cy5 fluorophores which can be simply quantified by single-molecule detection as a reflection of telomerase activity.Benefiting from the remarkable specificity of Taq DNA ligase-mediated ligation reaction,the robust signal output via multistage amplification cycles and the intrinsic high signal-to-noise ratio of single-molecule detection,this strategy exhibits high assay performance which can not only discriminate cancer cells from normal somatic cells but also quantify telomerase activity in as low as 1 He La cell.Moreover,the proposed method can be exploited to screen telomerase inhibitors,providing a feasible and powerful tool for clinical diagnosis and anticancer treatment.(2)We developed a fluorescent biosensor for single-molecule detection of FTO by integrating Maz F-mediated N6-methyladenosine(m6A)determination and poly(A)polymerase/DSN-assisted signal amplification.N6-methyladenosine(m6A)is the most prevalent internal modification in mammalian cells.The level of m6A is dynamically regulated by RNA demethylases[e.g.,FTO and Alk B homolog 5(ALKBH5)]and RNA methyltransferases such as methyltransferase-like 3(METTL3)and methyltransferase-like 14(METTL14).The imbalance of FTO expression is closely related to cancer and cardiovascular disease.Therefore,the quantitative analysis of FTO is of great significance for biomedical research and clinical diagnosis of diseases.In the presence of FTO,the m6A site of single-stranded RNA(ss RNA)substrate is removed,generating a demethylated ss RNA substrate.The demethylated ss RNA can be specifically cleaved by Maz F.After magnetic separation,the cleavage product with 3’-OH was amplified by poly(A)polymerase,producing long poly-A sequences.The resultant poly-A sequences can bind with poly-T-contained signal probe to form DNA/RNA double strand,inducing DSN-mediated cyclic cleavage of signal probe,producing an enhanced Cy5fluorescence signal.This method exhibits high sensitivity and specificity.It can screen potential inhibitors,quantify the level of intracellular FTO at the single cell level,accurately discriminate the FTO level in lung tissues between healthy persons and lung cancer patients,holding great potential in biomedical research and drug development.