BPS2026 – Linker architecture tunes fluorescence lifetime induction of Riboglow probes for RNA detection

The Riboglow platform consists of a short RNA sequence that binds a small molecule ligand (the probe), made up by an RNA-binding moiety and a fluorophore connected by a chemical linker. Probe binding to these short RNAs induces a significant fluorescence lifetime increase detectable via fluorescence lifetime imaging microscopy (FLIM). To optimize Riboglow as a robust RNA imaging platform, strong fluorescence contrast between free and RNA-bound probe is paramount. The chemical architecture of the probe’s linker segment affects fluorescence lifetime increase upon RNA binding. Here, we systematically designed a probe linker series where linkers are comprised of glycine repeats. Fluorescence lifetime measurements of these new probes demonstrate greater fluorescence contrast of glycine linkers over PEG linkers in the presence and absence of the RNA ligand at comparable linker lengths. Among the glycine linkers, the linker length was correlated with fluorescence lifetime induction, indicating FRET as a contributing mechanistic factor. Interestingly, the fluorescence lifetime induction of two probes differs significantly despite having similar lengths and only minor structural differences, highlighting the sensitivity of fluorescence lifetime to linker construction. These data, along with cellular lifetime and RNA binding analyses, underscore the importance of deliberate linker development for optimized fluorescence lifetime contrast in the Riboglow system. A mechanistic understanding of the fluorescence signal changes upon probe/RNA binding will guide rational designs of new generations of Riboglow probes.