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

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.