Label-Free Telomerase Detection in Single Cell Using a Five-Base Telomerase Product-Triggered Exponential Rolling Circle Amplification Strategy.

Abstract

Telomerase is a universal biomarker of malignant tumors. Sensitive and reliable analysis for telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Herein, a novel fluorescent strategy was proposed for sensitive and label-free detection of telomerase activity. One highlight of this strategy is that an exponential signal amplification can be triggered by a very short telomerase extension product (TEP). Without adding dATP, the designed telomerase primer can be easily controlled to extend five bases (GGGTT) to give short TEP with definite length. The resulting short TEP can then be constructed as a circular rolling circle amplification (RCA) template and thus initiate a nicking enzyme-mediated exponential RCA, producing G-rich amplification products that can be sensitively probed via specific binding between the fluorescent dye Thioflavin T (ThT) and the nucleic acid G-quadruplexes. Elevated telomerase translocation efficiency, combined with exponential signal amplification and specific probing of RCA products by ThT, endow the sensing platform with extraordinarily high detection sensitivity. The requirement for short TEP increases the possibility to analyze telomerase with low activity. The proposed sensing platform can achieve sensitive telomerase activity detection in individual cells, even with the interference of accumulated normal cells. It was also demonstrated to show excellent capability in screening for the inhibitors of telomerase. Therefore, the proposed sensing platform has great potential for not only clinical diagnosis but also anticancer drug development.