Highly sensitive detection of telomerase activity in tumor cells by cascade isothermal signal amplification based on three-way junction and base-stacking hybridization.

Herein, We report a simple and highly sensitive telomerase activity assay that integrates two consecutive isothermal signal amplification processes, namely, three-way junction triggered DNA-machine (3WJ-DNAM), and base-stacking hybridization assisted "biological circuit" DNA-machine (BSHBC-DNAM). In the presence of telomerase, the 3WJ are formed by the hybridization between the telomerase product and 3WJ-probes (3WJ-primer and 3WJ-template), which will initiate an autonomous 3WJ-DNAM by multiple processes of replication, nicking, and strand displacement, continuously generating short oligonucleotides as "triggers". These "triggers" will then provide additional stability for another two primers with a shared 5-bp complementary sequence at each 3'-end via base-stacking hybridization. And the BSHBC-DNAM are subsequently carried out by the strand-displacement induced circular utilization of "Trigger". Eventually, the single-stranded DNA (ssDNA) is generated in large quantities, and a significant fluorescence enhancement is observed due to the hybridization between the ssDNA and molecular beacons (MBs). In this way, per telomerase-mediated elongation event is efficiently and specifically converted into the greatly amplified fluorescence signals. This novel sensing strategy permits measurement of telomerase activity in cell extracts over the range of 3-5000 Hela cells, which is comparable or even superior to most previously reported methods. Using somatic and tumor cell lines, the selectivity and generality of the assay are investigated with satisfactory results. Furthermore, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) is also investigated, indicating its excellent performance in telomerase inhibitor screening.