BPS2026 – Modulating the fluorescent lifetime of Riboglow RNA tags through SELEX-driven mutations

Ribonucleic acids (RNAs) are one of the most fundamental biological macromolecules across all domains of life. They can transition genetic information when mRNAs are translated into proteins, or they can be noncoding and possess other functional properties, such as gene regulation. RNA functions are closely tied to their subcellular localization. Therefore, visualization tools are necessary to investigate and understand RNA functions both live and over time. Various fluorescence tagging and tracking systems have been developed. However, visualizing model RNAs in their complex environment relative to other RNAs and macromolecules is currently not possible. Multiplexed RNA imaging tools are urgently needed as RNAs often interact with each other and other biomolecules for correct functioning, and incorrect interaction can lead to disease states. We previously developed a fluorescence lifetime imaging microscopy (FLIM)-based tagging platform, Riboglow-FLIM, consisting of a genetically encoded RNA tag that binds a fluorescent probe, causing an increase in fluorescence intensity and fluorescence lifetime. Interestingly, altering the RNA tag sequence while keeping the fluorescent probe consistent results in varying fluorescent signals (such as fluorescent lifetime), which can be differentiated. We found that combining rational RNA sequence design and mutational analysis enables the identification of RNA variants with greater fluorescence signal differentiation. Then, we used unbiased sequence library expansion through SELEX for further RNA tag sequence space exploration. We coupled magnetic beads to our probe’s binding moiety to select for RNA tags that bind our probe. We measured probe fluorescence lifetimes to understand the relation between the sequence space and the fluorescent signal, allowing the development of more RNA tags. We plan to use these RNA tags in live cells to benchmark a suite of RNA tags to use simultaneously and to understand the dynamics of RNAs that interact.