Templated 3' terminal fluorescent labeling of RNA using Klenow DNA polymerase.

ABSTRACT: A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3' end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs. In this method:•Klenow DNA polymerase adds an amino-modified nucleotide to the 3' end of the RNA, guided by the DNA oligonucleotide template.•An NHS-ester dye is then conjugated to the amino-modified RNA, forming a covalent amide bond.•For highly structured RNAs, DNA oligonucleotides complementary to the RNA disrupt pre-existing intramolecular RNA structur

SUBMITTER: Mwangi MN 

PROVIDER: S-EPMC11407071 | biostudies-literature | 2024 Dec

REPOSITORIES: biostudies-literature