Detection And Imaging Of Telomerase And MiRNA Based On Novel Enzyme-free Biosensor System

Telomere is an important DNA-protein composite structure that constitutes the end of linear chromosomes in eukaryotes.It plays a vital role in maintaining the integrity of the genome and the stability of chromosomes.Telomerase is a reverse transcriptase that can maintain the length and activity of telomeres.Telomeres and telomerase are essential in life processes such as cell aging,cancer development,and maintenance of stem cell totipotent self-renewal ability.The imbalance between them is likely to induce cancer or other diseases.Telomerase is active in most cancer cells,maintaining the length and structure of telomeres,making cancer cells immortalized.Telomerase is an important cancer marker,which can be used as a basis for cancer diagnosis and treatment,and its detection has a wide range of applications.Based on the basic principles of telomerase amplification,this paper designs corresponding DNA nanostructures and recognition probes,and builds signal amplification system based on functionalized nanomaterials.We use electrochemical and fluorescent technologies as key detection methods,developing ultrasensitive biosensors for quantitative detection and intracellular imaging research related to telomerase activity.The details are as follows:1.The paper describes a voltammetric method for the quantitation of the activity of telomerase extracted from cancer cells.A thiolated single-stranded telomerase substrate primer was firstly immobilized on a gold electrode.In the presence of a mixture of telomerase and deoxynucleotide triphosphates,the primer becomes elongated and contains repetitive nucleotide sequences(TTAGGG)n.After hybridization with blocker DNA,gold nanoparticles are added and captured by the elongated singlestranded DNA.This reduces the charge transfer resistance of the gold electrode.The telomerase activity is then quantified via differential pulse voltammetry,typically at 0.12 V(vs.SCE).The method is PCR-free,rapid,and convenient.It was applied to the detection of HeLa cells via the telomerase activity of lysed cells.The detection range was from 20 to 2,000 cells and the detection limit was as low as 20 cells.2.We fabricated a ratiometric electrochemical strategy for the ultrasensitive analysis of telomerase activity.The hairpin DNA probe labeled with methylene blue(MB)tags at its 5' termini is firstly immobilized on the gold electrode.It then hybridizes with the telomerase substrate primer labeled with ferrocene(Fc)tags.With the telomerase-catalyzed reaction,a repeating sequence of(TTAGGG)n is generated,which is able to trigger the conformational transduction of the hairpin DNA probe.Consequently,MB tags are released from the electrode surface and the oxidation peak current(IMB)decreases.Meanwhile,the Fc signal remains stable,owing to the DNA conformational change,and the peak current(IFc)can be used as the internal control,which improves the signal stability.This smart design allows reliable target recognition and the obtained results assure excellent analytical performances.IMB/IFc is found to be linearly related with logarithmic telomerase concentration with a quite low limit of detection.This proposed ratiometric method may emerge as a powerful practical tool for the analysis of telomerase activity,which may also be developed as a versatile tool for other nucleic acid assays.3.Telomerase catalyzes the elongation of telomeres,which is closely associated with tumorigenesis.Therefore,development of reliable and convenient methods for telomerase activity analysis is critical for clinical diagnosis of cancers.However,most of the current probes for intracellular telomerase activity assay suffffer from inevitable false-positive interferences.In this study,we have proposed a novel fluorescence resonance energy transfer(FRET)-based strategy for ultrasensitive monitoring intracellular telomerase activity with an amplification-free procedure.DNA nanoprobes constructed using a DNA tetrahedron and Flare DNA are designed to circumvent the problem of false-positive interference.Generally,telomerase catalyzes DNA extension and changes the configuration of the DNA nanoprobe,which finally leads to the increase of the distance between two labelled fluorophores.Based on the notably decreased FRET effiffifficiency,a simple ratiometric sensor is established for the detection of telomerase activity at the single-cell level,which is much suitable for clinical applications.In addition,this method can be further applied in the detection of telomerase inhibitors,which is important for anti-cancer drug discovery.4.Telomerase and miRNA are overexpressed in tumor cells and have been recognized as biomarkers for tumor detection.Here,we designed a DNAzyme walking device triggered by target miRNA,combining with AuNPs probe structure for telomerase activity sensing.Through the chain replacement reaction,the target miRNA opens two hairpin structures and circularly amplified to form a bipedal DNAzyme walker.In the AND logic gate(scheme A),the presence of telomerase(Te)causes TP primers to be prolonged,and then bind to the locking chain.The bipedal DNA enzyme and Mn2+catalyzed reaction,and the carboxyl fluorescein(FAM)inhibited by AuNPs was released into the solution for fluorescence restoration.In the absence of either or both of the two targets,the fluorescence of the detection system cannot be recovered.In the OR logic gate(scheme B),the bipedal DNAzyme could independently open the hairpin structure on the AuNPs probe and release the FAM signal facilitated by Mn2+.The telomerase extended product(TPE)can hybrid with FAM locking chain and released into the solution to produce signal.In the presence of single target,the fluorescence signal can be recovered,while in the presence of two targets,the fluorescence intensity increases.In addition,the AuNPs probe complex containing signal molecules,and manganese dioxide(MnO2)nanosheets loaded with DNAzymes and TP primers,are available for cellular uptake for intracellular imaging.These method use biological logic gate and DNAzyme walkers,greatly improving the applicability and sensitivity of the probe detection,and have been successfully applied in vitro detection of telomerase and miRNA as well as the intracellular imaging research,which is of great significance for clinical diagnosis.5.This study strategically fabricates multifunctional nanostructure to allow the ultrasensitive detection of telomerase and miR-21 simultaneously and the corresponding intracellular bioimaging.The TDNs-Flare probe,which functionalized with gold nanoparticles(AuNPs)modified tetrahedral DNA nanostructure(TDNs)as the dual functionalized substrates,are composed of Flarel(Cy5)and Flare2(CDg)as different responsive reporters.In the presence of the targets,incomplete complementary DNA chains are competitively replaced,resulting in the simultaneous recovery of the dual fluorescent signals.The CDg fluorescence has a linear range with miR-21 from 1 fmol to 100 pmol,whereas the Cy5 fluorescence accomplishes sensitive telomerase activity detection down to 10 HeLa cells with a dynamic range from 10 to 5000 HeLa cells.This study demonstrates that the TDNs-Flare probe can be an excellent choice for the quantitative determination of cancer biomarkers,and is successfully used for responsive bioimaging.Moreover,the nanostructure not only overcome the obstacles in the simultaneous detection of multiple biomarkers at the cellular level but also have promising potential in clinical diagnoses.