Dynamic-Responsive DNA Tetrahedral Probes Are Design Ed For In Situ Analysis Of Telomerase In Living Cells

Telomerase is a ribonucleoprotease complex,its catalytic core includes telomerase reverse transcriptase(TERT)and telomerase RNA(TER),which can catalyze the addition of telomerase repeat sequence(TAAGGG)n to the end of telomeres.The different structure in the RNA and protein components of telomerase can regulate the processive ability of telomerase,and the processive ability of telomerase plays a key role in maintaining telomere steady state and telomere length.Telomerase function is the basis of cell proliferation and differentiation,and is directly related to various diseases such as cancer and genetic diseases.Therefore,accurate and sensitive detection of telomerase is of great value for the early diagnosis of diseases and the evaluation of drug effect.DNA nanostructures have been of great interest to researchers in analysis and testing owing to their editability and biocompatibility.Among them,DNA tetrahedral nanomaterials can be synthesized by a simple self-assembly method,and have the advantages of strong stability,low cytotoxicity,easy modification of structural sites,and easy endocytosis of the nanometer size.In recent years,DNA tetrahedrons have been widely applied as carriers in the field of biomedicine for in-situ imaging,biosensing and gene therapy.Based on the above analysis,biological probes based on functionalized DNA tetrahedron was constructed,which realize the sensitive and accurate detection of telomerase and uses it for cell imaging.This article is divided into three chapters:Chapter 1 is an introduction,which mainly summarizes telomere and reaction mechanism,research significance of telomerase currently.Meanwhile,we introduce the traditional and new detection methods that have been reported for telomerase,and the scientific problems and research contents mainly solved in this paper.In chapter 2,a sequentially lighting-up multicolor DNA tetrahedron nanoprobe(SLMN)for analyzing telomerase in living cells is developed.SLMN is composed of DNA tetrahedrons,a long DNA strand(LD),and three types of MBs(called MB 1,MB2,and MB3,respectively).MBs are respectively labeled with fluorescent groups FAM,Cy5,and HEX with different emission wavelengths.A telomerase substrate(TS)domain containing in LD strand,is extended with telomeric repeats under the action of telomerase.Owing to the product length limit and sequence complementarity,when repeat unit is added,it can only hybridize with the nearest molecular beacon.The extended telomere repeat sequence can sequentially intersect with fixed molecular beacons,and is accompanied by sequential light-up fluorescence.By resolving the signal,the contribution of a specific length product can be revealed,instead of obtaining a mixed signal for the entire product.This design can not only detect the activity of telomerase in the cell,but also realize the real-time monitoring of telomerase action on the substrate and the in-situ characterization of the product length distribution.In chapter 3,a DNA tetrahedron docking assembly(DTDA)for imaging telomerase activity exactly in tumor cells is developed.The DTDA bears a pair of bimolecular DNA triplexes in two of its vertexes,which are stabilized by Watson-Crick and Hoogsteen interaction under acid condition.DTDA maintains the structural integrity with the extracellular acid pH of tumor cells,but releases a homopyrimidinic,telomerase substrate-containing single-stranded DNA(HTS)from the triplex with the extracellular alkaline pH of non-cancerous cells.Once internalized into tumor cells,HTS becomes dissociated from DTDA into cytoplasm,due to the relatively high pH value inside cell.It then gets elongated by the intracellular telomerase,and docks to the other two vertexes via toehold-mediated strand displacement,returning to the DTDA after its separation and accompanied by fluorescence enhancement.As for non-cancerous cells,HTS does not enter into cells,resulting in no subsequent intracellular docking event.DTDA can precisely distinguish tumor cells from non-cancerous cells in which telomerase are both expressed.The strategy can offer a new paradigm for studying biomarker in targeted cells.