A New Method For Detection Of Telomerase Activity In Living Cells Based On Hybridization Chain Reaction

Telomerase activity is highly increased in tumor cells compared with normal human tissue cells,which is related to cellular immortalization and tumor progression.However,one of the most common methods for telomerase detection,polymerase chain reaction(PCR)-based telomeric repeat amplification protocol(TRAP),is complicated and the activity of Taq polymerase used in PCR is easily inhibited by cell extracts.With the development of materials science,many biosensing methods are used for the detection of telomerase activity in cells,but there are also problems with low sensitivity and specificity.In view of the above problems,this study developed a new method for the detection of intracellular telomerase activity based on hybridization chain reaction(HCR).The specific contents are as follows:The telomerase-targeting responder-transmitter DNA complex(HPT)consisting of telomerase primer sequence(HP)and a HCR initiator(trigger)is transfected into cell plasma by liposome.In the presence of telomerase,HPT can be recognized and extended,producing plenty of triggers which initiate HCR amplification reaction.Finally,a long nicked dsDNA with a lot of outstretched single chains is formed by hybridizing with Q of the reporter complex,generating an enhanced fluorescence signal.In cell lysate,the method was proved to be feasible by gel electrophoresis and fluorescence analysis,and a series of reaction conditions were optimized.Telomerase activity in cell lysate was analyzed under optimal reaction conditions,and the detection limit was 578 cells/100 ?L.When transfected by liposome nanoprobes,obvious fluorescence signal could be seen in HeLa cells.We used ASODN specifically inhibit telomerase activity in HeLa cells,and then detected the telomerase activity,fluorescence signal inside cells decreased significantly,indicating that this method can be used for sensitive and specific detection of telomerase activity in living cells.By detecting and comparing telomerase activity in different cells,this method is expected to be an auxiliary diagnosis method of cancer cells with the characteristics of increased telomerase activity.