A New Method For Detecting Telomerase Activity Was Studied

Telomerase can synthetize telomeric sequences by using its RNA as template to ensure the stability of telomeres length and function,resulting in cell immortalization.Therefore,telomerase was considered as a crucial tumor marker and therapy target.It is importantly significant to sensitively and reliably detect telomerase activity.Considering that,the dissertation puts forword simply and reliably colorimetric methods of telomerase activity,aiming at achieving convenient,low-cost and visual detection of telomerase activity.The contents of the dissertation include the following three sections.1.Research on DNAzyme-based colorimetric assay for visual detection of telomerase activityG-quadruplex DNAzyme,a type of artificial enzyme which consis,ts of G-rich single-stranded DNA and hemin(iron(III)-protoporphyrin ?),can effective catalyze 3,3',5,5'-tetramethylbenzidine(TMB)/H2O2 oxidation reaction,achieving visual detection.Herein,we developed a reliable and facile colorimetric method of assaying telomerase activity by combining catalysis ability of G-quadruplex DNAzyme and self-amplification of telomerase reaction.MB-TS complex was prepared by conjugating TS primer on MBs.A long G-rich single-strand DNA with TTAGGG as units would form in the presence of telomerase and dNTPs.MB/elongated TS complex was magnetic separated from telomerase reaction solution.In the presence of K+ and hemin,G-rich telomere DNA formed G-quadruplex DNAzyme and TMB/H2O2 oxidation reaction was efficiently catalyzed by the DNAzyme,realizing colorimetric assay for telomerase activity.One TS could be elongated numerious TTAGGG repeat units and multiple DNAzymes units formed in the presence of K+ and hemin,achieving signal amplification.The use of MBs may avoid interfaces from cell extract and enrich products of telomerase reaction,leading to a high signal-to-noise ratio.Telomerase activity equivalent to 20 and 10 HeLa cells could be determined using naked eyes and UV-vis spectrophotometer respectively.In healthy human serum matrix,real telomerase activity of 100 HeLa cells was visually detected by naked eye.2.Research on sensitive colorimetric assay for visual detection of telomerase activityBased on self-amplification of telomerase and magnetic enrichment,a reliable and ultra-sensitive colorimetric POC detection of telomerase activity was developed.Telomerase substrate(TS)primer was assembled on the surface of magnetic beads(MBs)by CHO/NH2 reaction.In the presence of telomerase,multiple repeats of TTAGGG would be added on 3' end of TS.The elongated telomere sequences could hybridize with biotin modified complementary DNA(cDNA)and further bound with catalyst horseradish peroxidase(HRP).By magnetic separation,redundant cDNA and HRP were discarded.Specific bound HRP effectively catalyze H2O2-mediated oxidation reaction of TMB,transforming telomerase activity into colorimetric output and realizing colorimetric detection by naked eyes.Telomerase activity equivalent to 5 HeLa cells and 1 HeLa cell can be reliably detected with naked eyes and UV-vis spectroscopy respectively.Real telomerase activity 20 HeLa cells and 5 HeLa cells can be reliably detected with naked eyes and UV-vis spectroscopy respectively.In healthy human serum matrix,telomerase activity equivalent to 50 HeLa cells and 10 HeLa cells was reliably detected by naked eye and UV-vis spectroscopy respectively.3.A sensitive and real-time assay of restriction endonuclease activity and inhibition based on photo-induced electron transferHerein,a facile,sensitive and real-time fluorescent assay was proposed for studying restriction endonuclease activity and inhibition based on photo-induced electron transfer(PIET).A 3'-termini carboxyfluorescein(FAM)labled single-stranded DNA served as the signal probe.Single-stranded DNA complementary to signal probe contained three G bases at 5'-termini and these three G bases served as signal transforming element.Signal probe hybridized with its complementary DNA to form duplex substrate of endonuclease.In DNA duplex,FAM approached to G bases and the fluorescence of FAM was quenched based on PIET.In the presence of endonucleases,the specific cleavage of DNA substrate by endonucleases occurred,leading to separation of FAM from guanosines and fluorescent recovery.The enhanced fluorescence had a good linear relationship with the logarithm of EcoRI concentration over the range of 5×10-5-1×10-2 U/?L.The detection limit estimated as 3 times the standard deviation of blank signals was 4.0×10-5 U/?L,which was more sensitive than most previously reported methods.Moreover,real time monitoring of restriction endonuclease digestion process was faciely achieved.Inhibitor of endonucleases was screened easily as well.Therefore,it offers a novel simple,sensitive and real-time assay for protein-DNA interaction reseaches.