Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background

摘要

Genomic DNA damage and repair are involved in multiple fundamental biological processes, including metabolism, disease, and aging. Inspired by the natural repair mechanism in vivo, we demonstrate for the first time the construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background. The presence of DNA glycosylase can catalyze the excision repair of the damaged base, successively autostarting the self-directed replication through recycling polymerization extension and strand-displacement DNA synthesis for the generation of exponentially amplified dsDNAs. The resultant dsDNA products can be label-free and real-time monitored with SYBR Green I as the fluorescent indicator. Owing to the high efficiency of self-directed exponential replication and the absolute zero background resulting from the efficient inhibition of nonspecific amplification induced by multiple primer-dependent amplification, this strategy exhibits high sensitivity with a detection limit of 1 x 10(-8) U mu L(-1)in vitro and 1 cell in vivo, and it can be further used to screen inhibitors, quantify DNA glycosylase from diverse cancer cells, and even monitor various repair enzymes by simply changing the specific damaged base in the DNA template. Importantly, this assay can be performed in a label-free, real-time and isothermal manner with the involvement of only a single type of polymerase, providing a simple, robust and universal platform for repair enzyme-related biomedical research and clinical therapeutics.