Intracellular Detection Of Telomerase Activity And Telomerase RNA Ratio Fluorescence Method

Telomerase is a nuclear protein reverse transcriptase.It can synthesize telomere repeats TTAGGG at the 3' end of telomeric DNA using its own telomerase RNA as a template.Studies have shown that telomerase activity is up-regulated in 85%tumor cells,whereas is not detected in normal somatic cells.Therefore,telomerase is considered as an important tumor marker.Telomerase RNA is an important component of telomerase,and its expression level is directly related to telomerase activity.Therefore,telomerase RNA is considered to be a tumor marker.At present,researchers have successfully established a variety of methods for detecting telomerase activity,which can detect telomerase activity sensitively and reliably.Most of methods have been proposed to detect telomerase activity in cell extracts,which did not directly reflect the activity of telomerase in complex physiological environment.However,the extraction and storage process of telomerase may affect the telomerase activity.Therefore,intracellular imaging is more reliable to study the expression levels of telomerase and telomerase RNA in complex physiological environments.This is of great significance for the reliable analysis of tumor markers at the cellular level and the early diagnosis of disease.In this thesis,we developed fluorescence methods for detection of intracellular telomerase RNA and telomerase activity by combing strand displacement reaction with ratiometric fluorescence resonance energy transfer technology.The main research contents and results can be summarized into the following two parts:1.Ratiometric fluorescence method for sensitive detection of intracellular telomerase RNAA simple,sensitive and reliable ratiometric fluorescence resonance energy transfer method for detecting human telomere RNA(hTR)was developed based on strand displacement reaction.Carboxyfluorescein labelled hairpin probe(FAM-H1)and tetramethyl rhodamine labelled hairpin probe(TAMRA-H2)were transported into cells using MnO2 nanosheet as carriers.After entering cells,MnO2 nanosheet could be decomposed by glutathione and release the probes.In the presence of hTR,the strand displacement reaction between two fluorophore hairpin probes was triggered to form DNA duplexes and displace the hTR.The re-released hTR triggered another cycle of SDR.Such cyclic strand displacement reaction can continuously form FAM-H1/TAMRA-H2,which make the distance between FAM and TAMRA close to each other and produce fluorescence resonance energy transfer phenomenon.For extracellular detection of hTR,the change of fluorescence ratio was linearly responded to the concentration of hTR in the range between 0.1 nM and 5 nM with a detection limit of 77 pM.Fluorescence imaging analysis showed that the method could reliably reflect the expression level of hTR in cells,and could distinguish the difference of hTR expression between tumor cells and normal cells.Therefore,this method is a simple,sensitive and reliable ratiometric fluorescence method for detecting intracellular hTR.2.Ratiometric fluorescence method for sensitive detection of intracellular telomerase activityBased on the double signal amplification strategy of telomerase self-amplification and strand displacement reaction,a simple,sensitive and reliable ratiometric fluorescence resonance energy transfer method was established for detecting intracellular telomere activity.Two hairpin DNAs labeled with carboxyfluorescein and tetramethyl rhodamine(FAM-H1,TAMRA-H2),auxiliary DNA(A-DNA/T-DNA)and telomere substrate(TS)were transported into cells by MnO2 nanosheet.When it enters the cell,it can be decomposed to Mn2+ by the glutathione to release the DNA probes.In the presence of telomerase,it can extend the TS substrate to form a telomerase DNA elongation products.Then,the products can open A-DNA/T-DNA to release numerous T-DNA achieving first step amplification.T-DNA can trigger the SDR between FAM-H1 and TAMRA-H2 to form lots of DNA duplexes achieving second step amplification.At the end of the DNA duplexes,FAM and TAMRA are close to each other,which results in fluorescence resonance energy transfer.According to the change of fluorescence ratio,the reliable and sensitive detection of telomerase activity can be realized.The telomerase activity equivalent to 20 HeLa cells can be detected.The expression level of telomerase activity in cancer cells and normal cells can be distinguished by fluorescence imaging experiments.Therefore,it provides a simple,sensitive,and reliable method for the detection of telomerase activity in living cells.