# Integrating PLOR and SPAAC Click Chemistry for Efficient Site-Specific Fluorescent Labeling of RNA

## TL;DR

This paper introduces ePLOR, a new method that combines PLOR and SPAAC click chemistry to more efficiently label RNA with fluorescent markers at specific sites.

## Contribution

The novel ePLOR method integrates SPAAC click chemistry with PLOR to overcome steric hindrance and improve RNA labeling efficiency.

## Key findings

- ePLOR achieves nearly 100% efficiency in SPAAC reactions on RNA.
- ePLOR labels RNA sites with 2–2.5 times higher efficiency than PLOR.
- ePLOR enables labeling across various structural regions of riboswitch RNAs.

## Abstract

Precisely fluorescently labeling specific nucleotide sites of RNA is critical for gaining insights into the structure and function of RNA through multiple fluorescence detection techniques. The position-selective labeling of RNA (PLOR) method provides a promising strategy to achieve this, wherein the fluorophore-modified NTPs can be co-transcriptionally introduced to specific sites of nascent RNA by using T7 RNA polymerase (T7 RNAP). However, due to steric hindrance limitations, the efficiency of T7 RNAP in recognizing and incorporating large fluorophore-modified NTPs into RNA is far from satisfactory. To overcome this challenge, in this work, we developed an efficient PLOR variant (ePLOR) for the site-specific fluorescent labeling of RNA by integrating PLOR with a post-transcriptional SPAAC (strain-promoted azido-alkyne cycloaddition) click chemistry reaction. The efficiency of the SPAAC reaction occurring on RNA is nearly 100%. Consequently, ePLOR enables the precise fluorescent labeling of designated sites across various structural regions of SAM-VI riboswitch and adenine riboswitch RNA, with labeling and synthesis efficiencies that are 2–2.5 times higher than those of PLOR. The strategy developed in this work can be used for the efficient synthesis of a broader spectrum of long-strand RNAs with site-specific fluorescent labeling and greatly facilitate the detection of the structure and function of these RNAs.