In Situ Imaging Of Telomerase Activity In Living Cells

Telomerase,the telomere-specific reverse transcriptase,is a ribonucleoprotein complex consisting of template RNA and protein.This holoenzyme allows the maintenance of telomere length by adding tandem telomeric DNA repeats,(TTAGGG)n,at the ends of chromosomes.In normal human tissues,telomerase activity is inhibited,whereas in 85%of tumors,telomerase activity is reactivated,thus contributing to cellular immortalization and tumor progression.Therefore,analysis of telomerase activity plays a crucial role in development of novel tumor diagnostic strategies and screening of drugs.The predominant approaches for telomerase detection are based on telomere repeat amplification protocol(TRAP),which use cell extract to obtain telomeric repeat segments followed by diffe rent detection techniques.Recently,considering that acquiring the spatiotemporal variation of telomerase activity in living cells can provide insights into the biological mechanism study,the focus has been shifted to developing in situ methods for tracking telomerase activity.In this field,a few strategies have been reported by our groupand others,which mainly relied on monitoring the signals from conformational changes of DNA probes due to telomerase-catalysed primer elongation.However,methods with enhanced sensitivity for in situ imaging of telomerase are largely lacking due to the difficulty of implementing signal amplification inside living cells.Inspired by the DNA nature of telomerase primer,this problem can be solved by combining the biological function of telomerase with isothermal nucleic acid-based signal amplification.The specificity and predictability of nucleic acid base-pairing provide a rich design space for engineering amplification systems.For example,programmable hybridization chain reaction and catalyzed hairpin assembly(CHA)have been used in intracellular microRNA and mRNAimaging for enhancement of sensitivity.However,to the best of our knowledge,there is no report on using nucleic acid-based signal amplification to facilitate the telomerase imaging inside living cells.The works of this thesisare as follows:1.A cascade amplification approach for visualization of telomerase activity in living cellsThis work developed a cascade approach for visualization of telomerase activity in living cells by linking telomerase-responsive primer extension with CHA-based signal amplification in series through a DNA transmitter(Scheme 1).Compared to existing few reports on in situ telomerase imaging with an equivalent reaction ratio(1:1),13a,b the proposed strategy achieved multiplied enhancement of signal outputs from one extension event(1:n)because of the recycling use of the transmitter,thus providing a powerful strategy for in situ tracking intracellular telomerase activity with accurate catalytic amplification.The proposed strategy relied on the design of a liposome nanoprobe which encapsulated two function modules.The first was a telomerase-targeting responder-transmitter DNA complex(HPT),a nicked hairpin DNA structure composed of a telomerase primer tagged segment(HP)and a transmitter segment(T)from 5' to 3' end that were separated by the nick.The 3'-end of HP along with T was designed to be complementary to the 5'-end of HP,forming the stem part of the hairpin.The sequence of HP was 5'-AACCCT AACCCT AACTCT GCTCGA CGGATT CCCCCCCCCC AATCCG TCGAGC AGAGTT-3',with the underlined part showing the telomerase primer that can be recognized by telomerase.The second module contained a pair of metastable DNA hairpin(HI,H2),and a hybridized reporter complex(R-FQ)with strands respectively labeled by a fluorophore(F,FAM in this work)and quencher(Q,Dabcyl in this work),thus constituting a catalyzed hairpin assembly(CHA)configuration.Using HeLa cells as the model,upon transfecting the nanoprobe into cytoplasm,the oligonucleotide probes could be released(Scheme 1).In the presence of telomerase and dNTPs,the 3'end of HP could extend to produce telomeric repeated sequence which was just complementary to 5' end stem,leading to the displacement of T.The released T could act as the initiator and catalyzer of CHA recycling amplification by hybridizing to the exposed toehold domain of H1.The produced Hl-T dup lex promoted the generation of H1-H2 duplex followed by the displacement of T for the next catalytic round.H1-H2 could subsequently hybridize with R-F of the reporter complex,leading to the removal of R-Q,thus lightening up the telomerase activity in living cells.Moreover,to ensure sensitive and selective long-term intracellular tracing in real time,locked nucleic acid(LNA)nucleotides were incorporated into the reporter complex,owing to their excellent thermal stability and enzymatic resistance capability in the cell cytoplasm.2.Visualizeddetection of telomere length scale in vivo by telomerase-responsible DNA-MSN nanoprobeA length ruler for the detection of telomerase activity in cells was designed based on porous silica spheres as the carrier.The aim of the experiment is to use the principle of superposition method to realize the detection of telomerase activity in situ.The proposed method is based on the three different ring sizes of the hairpin DNA probes(ring-s,ring-m,ring-1)to distinguish the different length of extended telomerase TS with different ring sizes.Due to the fluorescence signal of the different ring diameters hairpin DNA probe can be turned on by the differnt extent length of the DNA sequence,respectively.When the telomerase TS primer was extended by 1 telomere,hairpin DNA with ring-s can be opened.And when the TS primer extended by multiple telomeres,then ring-m or ring-1 could be opened.At last,using the method of lamination,the fluorescence results were overlapped and the TS primers of telomerase could be measured visually,so the DNA modified MSN probe is called the telomerase extension degree ruler.Fluorescein was embeded in nanoparticles.When the modified nanoparticles of hairpin DNA was transfected into the cell,the fluorescein can be used as an internal standard,by which hairpin DNA fluorescence signal can be quantified.Finally,the detection of telomerase extended scale can be realized.The specific measurement of telomerase activity in living cells using nanoparticles as the carrier was expected to make important progress in the study of the biological properties of human telomerase.