The Design Of Dual-colored Ratiometric-fluorescent Oligonucleotide Probe And Its Applications In Biological Analysis

As a ribonucleoprotein enzyme,telomerase plays a vital role in catalyzing the synthesis of telomeric repeats of eukaryotic chromosomal ends to extend the length of telomere and keep cells immortal.Telomerase is a important tumor marker,and in recent years,it has undoubtedly become the focus of research.According to whether there are detectable telomerase activities in tissue cells,it can be determined whether cells are cancerous or not.Detection methods of telomerase are quite mature in vitro and in vivo,and there are still a lot of reports in the exploration of new means for detecting telomerase.Human telomerase RNA(hTR)is one of components of telomerase,acting as a reverse transcription template for telomerase catalytic reactions.The hTR is common in both normal and tumor cells,and the activity of hTR is more closely related with tumor grade in comparsion with the activity of telomerase.However,the passion of researchers for detecting hTR is less than detecting telomerase.Fluorescence means is a simple and quick method for assaying nucleic acids,but there are few methods that have a strategy to avoid false positives.Therefore,it is the focus of this paper to find a strategy to detect human telomerase RNA with high efficiency,convenience,stable and reliable signal.In this paper,we designed a dual-colored ratiometric-fluorescent oligonucleotide(DRO)probe based on the principle of fluorescence resonance energy transfer(FRET)to realize the quantitative assay of hTR.The main structure of this paper is as follows:In chapter one,we mainly reviewed telomerase and hTR,and enumerated some of current classic methods for detection of both telomerase and hTR.At the same time,we briefly introduced the principle of FRET and its application in chemistry and biology.In chapter two,discussion of the design and detection principle of the DRO probe.We reported the synthesis of DRO probe,and study and discussed the characteristics of signal stability,selectivity and reversibility of the probe.The detection principle of DRO probe was verified by gel electrophoresis.The results showed that the probe has good signal stability,high specific selectivity to the target and even "naked eye" color variation in the detection process.At last,we find that the probe also showed a good effect for analyzing hTR in cell extracts.In chapter three,we introduced several kinds of synthetic methods of mesoporous silica and its surface modification,and synthetic methods of gold nanoparticles,silver-coated gold nanoparticles,silica-coated gold nanoparticles,Fe304 magnetic nanoparticles.And all of these nanoparticles have the same property of surface adsorption of single-stranded oligonucleotides.Moreover,we can design some probe for detection of telomerase and hTR based on this property,and this is expected to provide an alternative strategy to future research.