title: Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.
abstract: Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
authors.forename: Xin
authors.identifier: 0000-0002-6629-9856
authors.initials: X
authors.lastname: Su
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
authors.forename: Zehao
authors.initials: Z
authors.lastname: Li
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
authors.forename: Xinzhong
authors.initials: X
authors.lastname: Yan
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University , Changsha, Hunan 410081, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wei
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University , Changsha, Hunan 410081, China.|College of Chemistry, Nankai University , Tianjin 300071, China.
authors.forename: Lehui
authors.identifier: 0000-0003-0522-2342
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
authors.forename: Changyuan
authors.initials: C
authors.lastname: Yu
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008856:Microscopy, Fluorescence; D010316:Particle Size; D000072760:Single Molecule Imaging; D013269:Stochastic Processes; D013499:Surface Properties; D019098:Telomerase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D004247:DNA; D019098:Telomerase