# Templated 3ʹ terminal fluorescent labeling of RNA using Klenow DNA polymerase

## TL;DR

A new method uses Klenow DNA polymerase to precisely label the 3ʹ end of RNA molecules with fluorophores, even for structured RNAs.

## Contribution

A chemo-enzymatic method for 3ʹ terminal RNA labeling that works on structured RNAs of any length.

## Key findings

- Klenow DNA polymerase adds an amino-modified nucleotide to the RNA's 3ʹ end using a DNA template.
- NHS-ester dyes form covalent bonds with the modified RNA, enabling fluorescent labeling.
- Complementary DNA oligos disrupt RNA structures, enabling labeling of highly structured RNAs.

## 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 3ʹ 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 3ʹ 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 structures.

Klenow DNA polymerase adds an amino-modified nucleotide to the 3ʹ 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 structures.

This methodological advancement enables site-specific incorporation of a single modified nucleotide at the 3′ terminus of various RNA substrates, irrespective of their length or secondary structure. The user-friendly nature of the technique, with minimal modifications required for different RNA targets, makes it readily adaptable by a broad range of researchers. This approach has the potential to significantly improve the development of functionalized RNA for various applications.

Image, graphical abstract