Development of fluorescence-based enzyme activity detection technologies

Abstract

Enzymes as a biocatalyst, play important roles in biological processes including DNA replication, DNA recombination, and transcription and in the fields of biotechnology including polymerase chain reaction, molecular cloning, and DNA sequencing. Their altered levels have served as an indicator for a number of pathological conditions and recognized as attractive therapeutic targets for the treatment of diseases such as cancers and AIDS. Due to their biological and medical significance, a wide variety of detection strategies have been developed for the enzyme activities. In particular, the fluorescent strategies have garnered the special attention due to their advantageous features including high sensitivity, reliability, and simplicity. However, there still remain the drawbacks to be overcome including high-cost and complexity coming from the labeling with fluorophore and quencher and complicated detection procedures. In addition, the sensitivity needs to be further improved. In this dissertation, we have developed novel fluorescent sensing strategies for the enzyme activities by employing unique features of fluorescent nucleobase analogs, nanocomposites, and isothermal amplification methods. Based on this, the activities of base excision repair (BER) enzymes, nucleases, and phosphatases were successfully analyzed, overcoming the above-mentioned limitations of previous methods.