Study On The New Methods For Biomolecules Detection Based On DNA-Templated Copper Clusters

In recent years, metal cluster has obtained more attention in research and application as an emerging probe. Comparing with organic fluorescent dyes, metal cluster shows superior fluorescence property and broad prospects in biosensing and biology imaging. For example, good biocompatibility, large stokes shift and high fluorescence quantum yield. This paper explored the application of DNA-templated copper cluster in biological chemistry as follows.Firstly, this dissertation designed a sensitive method for coenzymeⅠdetection based on dumbbell-shaped DNA-templated copper clusters. Dumbbell-shaped DNA could be elongated to form a long double strand product by Klenow Fragment polymerase, inducing strong fluorescent signal. In the presence of coenzymeⅠ, the E.Coli DNA ligase would catalyze the ligation of 5’ terminal phosphate group and 3’terminal hydroxy, resulting weak fluorescent signal. This novel assay can detect coenzymeⅠ as low as 0.2 nM with high selectivity against other coenzymeⅠanalogs, and also can analyze coenzymeⅠ in biological samples.Secondly, this dissertation developed a novel label-free fluorescent assay for detecting T4 polynucleotide kinase/phosphatase activity and its inhibitor screening based on poly(thymine)-templated fluorescent copper cluster. The probe has a tail consisting of poly(thymine) bases, which could be used for the template of copper clusters preparation. The phosphorylated hairpin probe could be elongated to build a long strand product by Klenow Fragment polymerase only in presence of T4 polynucleotide kinase/phosphatase, which prohibited the preparation of copper clusters and induced low fluorescent signal. The detection limit of this method is 0.25U/mL. T4 polynucleotide kinase/phosphatase activity was also analyzed in biological environment with satisfactory results.Thirdly, this dissertation proposed a simple, sensitive, low cost and label-free method for exonuclease Ⅲ detection by the poly-T loop templated copper clusters.The mechanism is mainly based on the poly(thymine) loop-dependent formation of copper clusters and the 3’ hydrolytic reaction of exonuclease Ⅲ. The exonuclease Ⅲcould degest the 3’ termini of hairpin probe stem, unfolding the poly-T loop with the decrease of fluorescence. And compared with the previous reported works, this strategy doesn’t need any complex DNA sequence design, fluorescence dye label, and sophisticated experimental techniques. In addition, it has great potential for exploring the application in life activity and medical diagnosis.