Multivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluated the impact of low, prebiotically-relevant polyion multivalency in coacervate performance as functional compartments. As model polyions, we used positively and negatively charged homopeptides with one to 100 residues, and adenosine mono-, di-, and triphosphate nucleotides.Polycation/polyanion pairs were tested for coacervation, and resulting membraneless compartments were analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the relatively shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes. Hence, reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, but they can offer advantages over higher molecular weight analogues.
The COVID-19 pandemic persists despite the development of effective vaccines. As such, it remains crucial to identify new targets for antiviral therapies. The causative virus of COVID-19, SARS-CoV-2, is a positive-sense RNA virus with RNA structures that could serve as therapeutic targets. One such RNA with established function is the frameshift stimulatory element (FSE), which promotes programmed ribosomal frameshifting. To accelerate identification of additional functional RNA elements, we introduce a novel computational approach termed the Functional RNA Identification (FRID) pipeline. The guiding principle of our pipeline, which uses established component programs as well as customized component programs, is that functional RNA elements have conserved secondary and pseudoknot structures that facilitate function. To assess the presence and conservation of putative functional RNA elements in SARS-CoV-2, we compared over 6,000 SARS-CoV-2 genomic isolates. We identified 22 functional RNA elements from the SARS-CoV-2 genome, 14 of which have conserved pseudoknots and serve as potential targets for small molecule or antisense oligonucleotide therapeutics. The FRID pipeline is general and can be applied to identify pseudoknotted RNAs for targeted therapeutics in genomes or transcriptomes from any virus or organism.
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. However, 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. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life.
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