tRNA structure-seq in peptide-rich droplets

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology, and has been proposed as a prebiotically-plausible way to accumulate RNA. Compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Transfer RNAs are essential for protein synthesis and predate the last universal common ancestor (LUCA) of all organisms, making them relevant to both extant and ancient biology. Since tRNAs are the most heavily modified RNAs known, with modifications crucial to RNA structure, function, and gene regulation, we also compared unmodified and naturally-modified tRNAs. We elucidate the effects of Mg2+ concentration, polyion charge ratio, and polyion length on tRNA structures. The approach herein, which can be applied to study other RNAs in diverse condensates, reveals that RNA can achieve native tertiary structure in a robust fashion in membraneless compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that modifications could have played a critical role in metabolic processes at the origin of life. We performed tRNA structure-seq (a type of dimethyl sulfate chemical probing followed by mutational profiling DMS-MaP-Seq) inside of peptide-rich droplets to study a T7 transcript of a single tRNA from S. cerevisiae to validate the the technique. Then, we purified natively modified tRNAs from E. coli and generated T7 transcripts of those same tRNAs and again performed tRNA structure-seq in peptide-rich droplets. For each type of RNA in each droplet condition, there were 3 independent control experiments and 3 independent DMS treatments performed.