Optimization of 3' adapter ligation for labelling RNA

Abstract

Fluorescent labelling of RNA is important for detecting and quantifying RNA. RNA with fluorophore added on either 5’ or 3’ end can be obtained by chemical synthesis but with a limitation of length. In this work, we demonstrate a labelling method based on ligation. A fluorescent labelled ssDNA adapter is ligated to the 3’ terminus of the RNA to give the fluorescent signal. We identify better conditions for the ligation. Factors including adapter types, PEG concentration, enzyme dosage, reaction time and RNA to adapter ratio were optimized. The ligation yield can reach to more than 60% under optimized conditions. Notably, this ligation based fluorescent labelling method would not significantly affect the biological properties of RNA, e.g. protein binding affinity, which provides a potential way for long RNA fluorescent labelling.