3’ Terminal end‐labeling of structured RNA by Klenow DNA polymerase

Abstract

RNA labeling is a key step in studying RNA localization, folding, and structural dynamics using fluorescence based assays. Current methods of fluorophore attachment to long (>100 nucleotides) and structured RNAs are hampered by low labeling yields and limitations in attachment position. These challenges have led to an interest in developing alternative methods that would enable the site‐specific labeling of long and structured RNAs. To this end, we have developed a site‐specific, chemo‐enzymatic method of fluorophore incorporation to RNAs. This novel method uses Klenow enzyme, a DNA polymerase, to add modified nucleotides to RNAs 3’ end in a templated manner. To facilitate this modified nucleotide addition in structured RNAs, we designed helper DNA oligonucleotides to disrupt any pre‐existing RNA intramolecular structure. Using this method, we have achieved efficient 3’ terminal end‐labeling of chemically synthesized and in‐vitro transcribed RNAs of different lengths with cyanine dyes.This method will be routinely used in fluorescence‐based assays that investigate the structure‐function relationships of RNAs involved in cancer and HIV progression.