Expanding the Riboglow-FLIM Toolbox with Different Fluorescence Lifetime-Producing RNA Tags

RNAs are essential elements of biology with subcellular localizations critical for function. Genetically tagging fluorescent RNA reporters to an RNA of interest allows for investigating RNA spatiotemporal dynamics. We previously developed a fluorescence lifetime imaging microscopy (FLIM)-based RNA-tagging platform, Riboglow-FLIM. Here, a genetically encoded RNA tag binds a fluorescent probe, causing an increase in both fluorescence intensity and fluorescence lifetime. Importantly, the Riboglow platform is derived from a bacterial riboswitch RNA family and different riboswitch sequences from nature may build the basis for multiplexing capabilities. We previously observed fluorescence lifetime differences for two RNA tags <i>in vitro</i> and in live mammalian cells as a proof-of-concept demonstration. As an in-depth expansion for multiplexing capabilities, here we evaluate the performance of different RNA sequences <i>in vitro</i> for systematically expanding the RNA tag sequence space of Riboglow-FLIM. We use two methods of varying the genetic tag to evaluate multiplexing capabilities, a literature-guided and a rational design approach. The literature-guided approach includes riboswitch sequences with both indirect and direct evidence of probe binding. For this, a phylogenetic tree of riboswitch-derived tags from indirect binding results was constructed, and RNA members from different branches were characterized. We also designed RNA mutations rationally based on insights from established Riboglow RNA tags. Together, nine different RNA tags yielded a wide range of fluorescence lifetimes for the Riboglow-FLIM platform, building the foundation to tag and track several different RNAs simultaneously. These findings will serve as the basis for achieving multiplexed RNA imaging in live cells using a fluorescence lifetime sensor.