RNA interactions are exceptionally strong and highly redundant. As such, nearly any two RNAs have the potential to interact with one another over relatively short stretches, especially at high RNA concentrations. This is especially true for pairs of RNAs that do not form strong self-structure. Such phenomenon can drive liquid-liquid phase separation, either solely from RNA-RNA interactions in the presence of divalent or organic cations, or in concert with proteins. RNA interactions can drive multimerization of RNA strands via both base pairing and tertiary interactions. In this article, we explore the tendency of RNA to form stable monomers, dimers, and higher order structures as a function of RNA length and sequence through a focus on the intrinsic thermodynamic, kinetic, and structural properties of RNA. The principles we discuss are independent of any specific type of biomolecular condensate, and thus widely applicable. We also speculate on how external conditions experienced by living organisms can influence formation of nonmembranous compartments, again focusing on the physical and structural properties of RNA. Plants, in particular, are subject to diverse abiotic stresses including extreme temperatures, drought, and salinity. These stresses and the cellular responses to them, including changes in the concentrations of small molecules such as polyamines, salts, and compatible solutes, have the potential to regulate condensate formation by melting or strengthening base pairing. Reversible condensate formation, perhaps including regulation by circadian rhythms, could impact biological processes in plants and other organisms.

Fragile X syndrome, the most common inherited form of intellectual disability, is caused by the CGG trinucleotide expansion in the 5’-untranslated region of the Fmr1 gene on the X chromosome, which silences the expression of the fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA, which encodes for the postsynaptic density protein 95, and together with microRNA-125a to mediate the reversible inhibition of the PSD-95 mRNA translation in neurons. The miR-125a binding site within the PSD-95 mRNA 3’-untranslated region (UTR) is embedded in a G-rich region bound by FMRP, which we have previously demonstrated folds into two parallel G-quadruplex structures. The FMRP regulation of PSD-95 mRNA translation is complex, being mediated by its phosphorylation. While the requirement for FMRP in the regulation of PSD-95 mRNA translation is clearly established, the exact mechanism by which this is achieved is not known. In this study, we have shown that both unphosphorylated FMRP and its phosphomimic FMRP S500D bind to the PSD-95 mRNA G-quadruplexes with high affinity, whereas only FMRP S500D binds to miR-125a. These results point towards a mechanism by which, depending on its phosphorylation status, FMRP acts as a switch that potentially controls the stability of the complex formed by the miR-125a-guided RNA induced silencing complex (RISC) and PSD-95 mRNA.

G-Quadruplexes (GQs) are compact, stable structures in DNA and RNA comprised of two or more tiers of quartets whose G-rich motif of tracts of two or more G's occurs commonly within genomes and transcriptomes. While thermodynamically stable in vitro, these structures remain difficult to study in vivo. One approach to understanding GQ in vivo behavior is to test whether conditions and molecules found in cells facilitate their folding. Polyamines are biogenic polycations that interact with RNA. Among common polyamines, spermine contains the highest charge and is found in eukaryotes, making it a good candidate for association with high-charge density nucleic acid structures like GQs. Using a variety of techniques, including ultraviolet-detected thermal denaturation, circular dichroism, size exclusion chromatography, and confocal microscopy, on an array of quadruplex sequence variants, we find that eukaryotic biological concentrations of spermine induce microaggregation of three-tiered G-rich sequences, but not of purely two-tiered structures, although higher spermine concentrations induce aggregation of even these. The formation of microaggregates can also be induced by addition of as little as a single G to a two-tiered structure; moreover, they form at biological temperatures, are sensitive to salt, and can form in the presence of at least some flanking sequence. Notably, GQ aggregation is not observed under prokaryotic-like conditions of no spermine and higher NaCl concentrations. The sequence, polyamine, and salt specificity of microaggregation reported herein have implications for the formation and stability of G-rich nucleic acid aggregates in vivo and for functional roles for understudied GQ sequences with only two quadruplex tiers.

A colorimetric nucleic acid based test for label-free pathogen detection has been developed and used for the detection of the Zika virus. The test relies on nucleic acid sequence-based amplification (NASBA) of a viral RNA followed by interrogation of the amplicon by a cascade of deoxyribozymes constituting a visual split deoxyribozyme (vsDz) probe. The probe consists of a split phosphodiesterase deoxyribozyme, which forms its catalytic core upon binding to a specific amplicon fragment. The catalytically active complex recognizes and cleaves an inhibited peroxidase-like deoxyribozyme (PDz), thereby activating it. Active PDz catalyzes hydrogen peroxide-mediated oxidation of a colorless substrate into a colored product, thereby generating a visible signal. Viral RNA (10 6 copies/mL or higher) triggers intense color within 2 hr. The test selectively differentiates between Zika and closely related dengue and West Nile viruses. The reported technology combines isothermal amplification and visual detection and therefore represents a basis for the future development of a cost-efficient and instrument-free method for point-of-care nucleic acid analysis.

Guanine-rich regions of DNA or RNA can form structures with two or more consecutive G-quartets called G-quadruplexes (GQ). Recent studies reveal the potential for these structures to aggregate in vitro. Here we report effects of in vivo concentrations of additives—amino acids, nucleotides, and crowding agents—on the structure and solution behavior of RNAs containing GQ-forming sequences. We found that cytosine nucleotides destabilize a model GQ structure at biological salt concentrations, while free amino acids and other nucleotides do not do so to a substantial degree. We also report that the tendency of folded GQs to form droplets or to aggregate depends on the nature of flanking sequence and the presence of additives. Notably, in the presence of biological amounts of polyamines flanking regions on the 5′-end of the RNA drive more droplet-like phase separation, while flanking regions on the 3′-end, as well as both the 5′- and 3′-ends, induce more condensed, granular structures. Finally, we provide an example of a biological sequence in the presence of polyamines and show that crowders such as PEG and dextran can selectively cause its phase separation. These findings have implications for the participation of GQS in LLPS in vivo.