# Evaluating Linker Architecture in RNA-Detecting Riboglow Probes and Effects on Fluorescence Turn-On

## TL;DR

This paper studies how the chemical linker in RNA-detecting Riboglow probes affects their fluorescence, showing that glycine-based linkers improve performance and enable RNA visualization in live cells.

## Contribution

The study reveals that glycine linker composition and length significantly enhance fluorescence turn-on and enable RNA multiplexing in Riboglow probes.

## Key findings

- Glycine linkers produce higher fluorescence turn-on than polyethylene glycol linkers of similar length.
- Increasing glycine residues in the linker enhances fluorescence turn-on upon RNA binding.
- Linker composition affects fluorescence lifetime contrast, enabling RNA multiplexing and live-cell RNA visualization.

## Abstract

Riboglow probes are small molecules where a synthetic
fluorophore
is connected to an RNA-binding moiety via a chemical linker. Upon
binding a short RNA sequence, probe fluorescence intensity and lifetime
increase. The fluorescence change is modulated by the architecture
of the chemical linker. Here, we systematically interrogated the linker
composition in a series of Riboglow probes and assessed fluorescence
properties. We found that glycine linkers result in higher fluorescence
turn-on compared to a polyethylene glycol linker of similar length.
When varying the length of the polyglycine linker, we found that increasing
the number of glycine residues led to more substantial fluorescence
turn-on upon RNA-ligand binding. Surprisingly, the composition of
the Riboglow chemical linker influences fluorescence lifetime contrast
when comparing probe binding to two different RNA ligands, a quality
necessary for RNA multiplexing. Finally, evaluating probe fluorescence
lifetimes in live mammalian cells demonstrated the ability of new
Riboglow probes to visualize RNAs live. Insights gained from the systematic
assessment of the linker’s architecture will dictate the rational
design of future fluorophore-quencher probe designs.