DNA Machine-based Fluorescence Biosensors For The Detection Of Telomerase Activity

Telomerase is a kind of ribonucleoprotein which can add telomere repeats on the end of chromosome to stabilize the length of telomere DNA.The enhancement of telomerase activity can lead to continued division even immortalization of cells and has been closely associated with multiple diseases,such as cancers.Telomerase is regarded as an important biomarkers of cancers and the sensitive detection of telomerase activity is of great significance in the early diagnosis of cancers.DNA machine is a kind of DNA device which can perform molecular dynamic mechanical function in the presence of the fuel.It has attracted much attention due to the controllable dynamics and the advantage in the study of molecular interaction.The application has derived multiple directions based on the controllable movement in nanometer level of DNA machine,such as signal amplified DNA machine and molecular switched DNA machine,which have promising application in the development of sensitive and specific biological sensing platforms.In view of the above analysis,we developed fluorescence sensing platforms based on two kinds of DNA machines for the sensitive and accurate detection of telomerase activity.The dissertation can be divided into three chapters as follows:The first chapter summarized the structures and functions of telomere and telomerase,the relationship between telomerase activity and cell aging and tumorigenesis,the main detection methods of telomerase activity and their limitations.In addition,we introduced the mechanism and the application progress of DNA machine.In the second chapter,a label-free molecular beacons-based cascade amplification DNA machine was developed for the sensitive detection of telomerase activity.This strategy produced lots of G-quadreplex structures by the cascade amplification DNA machine,which can be used for signal output with the telomerase extension product together,increasing the detection sensitivity of telomerase activity.Telomerase activity in HeLa cells extracts equivalent to 50 HeLa cells/mL was successfully measured with a linear range from 50 HeLa cells/mL to 2000 HeLa cells/mL,which was comparable or even superior to most previously reported methods.In addition,the strategy was also successfully used to assay the inhibition effect of an inhibitor of telomerase activity,verifying that the assay holds the potential to screen telomerase inhibitors.In the third chapter,a fluorescent strategy based on a DNA tweezer molecular switch was constructed for the ratiometric detection of telomerase activity.The DNA tweezer kept open initially,making the fluorescence donor and acceptor far away from each other and the FRET efficiency low.Telomerase extension product can close the tweezer and bring the fluorescence donor and acceptor into close proximity,enhancing the high FRET efficiency.The strategy can be used for the ratiometric measurement of telomerase activity through the change of the FRET response before and after the closure of the DNA tweezer,and we introduced the replacement strands which can reopen the DNA tweezer to study of the processivity of telomerase.In this chapter,the feasibility of the method was verified by gel electrophoresis and fluorescence method,and we researched the analytical performance for telomerase activity under the optimal conditions.