Design Of Telomerase-Specific Fluorescence Probes And Their Applications In Cancer Cell Detection

Cancer is a disease with high morbidity and mortality,the early detection of cancer can significantly save lives.Thus,a great deal of effort has been devoted to the exploration of new technologies to detect early signs of cancer.Cancer biomarkers contain a large range of biochemical entities,such as proteins,nucleic acids,enzyme,and small metabolites.They can be used for risk assessment,diagnosis,prognosis,and for the prediction of treatment efficacy.Identification of tumor cells versus normal ones has been a daunting challenge despite its significant impact on cancer diagnosis,treatment and prognosis.The fundamental difficulties,however,are the heterogeneity of tumor cells and the lack of truly specific and ideally universal cancer biomarkers.In the present dissertation,the mainly results were briefly summarized as following:(1)We report a design of the combination of telomerase-specific spherical nucleic acids(SNAs)and molecular beacons(MB),bypassing the specific genotypic and phenotypic features of different tumor cell types and directly going towards the key point of cancer hallmark,telomerase activity.Taking advantage of cell-entry behavior of SNAs,we immobilized telomerase-targeting molecular beacons onto AuNPs surface to prepare a series of probes.The molecular beacon is consisted of a long telomerase primer(TP)-carrying strand and a short fluorophore-modified DNA strand(FL-strand)that are pre-hybridized before immobilizing onto AuNPs surface via the robust Au-thiol linkage.In the presence of telomerase,the TP-carrying strand is recognized by the catalytic core of telomerase,and elongated with telomeric repeat TTAGGG,resulting in formation of a more thermodynamically stable DNA hairpin structure.The FL-strand is subsequently displaced into the solution,restoring its fluorescence for detection.(2)A telomerase-specific probe was designed and synthesized.In the presence of telomerase,the fluorophore-modified DNA is released into the solution,restoring its fluorescence for detection,readily achieves tumor cell detection across multiple platforms,ranging from solution-based assay,to single cell imaging and in vivo solid tumor imaging.We envision the SNAs probe will impact cancer diagnosis,therapeutic response assessment,and image-guided surgery.(3)We developed a general platform to discrimination of tumor cells and normal cells with different telomerase expression level by the naked eye,which could be achieved by designing a dual-colored dyes modified ratiometric fluorescent oligonucleotide probe for qualitative and quantitative detection of telomerase activityin cell lysates.This probe was consisted of a FAM modified telomerase primer(FAM-TP strand)and a Texas Red modified molecular beacon strand(Tesax Red-MB strand)which was pre-hybridized onto AuNPs surface via the robust Au-thiol linkage.Once the target telomerase is added,a color variation from green to red occured.In contrast to the traditional methods,the realization of telomerase assay using two dyes modified ratiometric fluorescent oligonucleotide probe is rarely reported.By using this ratiometric fluorescent probe,telomerase activity in cell lysates could be precisely detected.This study is expected to exhibit exciting opportunities in medical diagnostics and therapeutic response assessment.