Ultrasensitive Detection Of Intracellular Telomerase Activity

Telomerase is an enzyme that catalyzes the protein and RNA template that isolates DNA at the end of chromosomes,the immortalization of cells.Telomerase plays an important role in extending telomere,maintaining telomere stability,genome integrity,long-term cell activity and continuous proliferation.Studies have shown that telomerase activity is reduced or absent in normal human cells,but is highly activated in almost all types of cancer(more than 85%),making it an important biomarker for early cancer diagnosis and a potential therapeutic target for cancer treatment.Even though great efforts have been made to develop efficient and reliable telomerase assays,most of them suffer from complicated procedures and limited sensitivity.Herein,we develop a simple method for one-step and ultrasensitive detection of telomerase activity on the basis of telomerase-triggered strand displacement amplification(SDA)-mediated primer generation-rolling circle amplification(PG-RCA).The presence of telomerase may catalyze the extension of detection probe and subsequently induce SDA reaction to generate abundant single-stranded RCA primers.The resultant RCA primers can bind to the circular templates to trigger PG-RCA reaction,producing a large amount of amplification products which can be easily monitored in a label-free manner by using SYBR Gold as the fluorescence indicator.In comparison with the reported amplification-based methods,this assay enables one-step and one-tube detection of telomerase activity under absolutely isothermal conditions(35?)without the involvement of any washing and separation steps,greatly simplifying the experimental procedures and avoiding the cross contamination.This assay exhibits high sensitivity with a detection limit of as low as 3 cells and a wide dynamic range of 4 orders of magnitude from 10 to 105 cells,and it can be further applied for the screening of telomerase inhibitors and the discrimination of cancer cells from normal cells.This paper developed an ultra-sensitive detection method for telomerase in cancer cells based on primer-generating rolling circle amplification under absolute isothermal conditions.The experimental scheme is ingenious in design,the experimental steps are simple,the operation is simple and not time-consuming,and the one-tube one-step method is implemented to detect telomerase activity under isothermal conditions.The telomerase activity signal is convert into nucleic acid signal through the telomere enzyme initiation strand displacement amplification to achieve higher sensitivity.Ultra-sensitive detection of telomerase activity is not only beneficial to the screening of telomerase inhibitors,but also has potential application value in biomedical research and clinical disease diagnosis.