Long Tracts of Guanines Drive Aggregation of RNA G-Quadruplexes in the Presence of Spermine

Abstract

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.

RNA multimerization as an organizing force for liquid–liquid phase separation

“…Such condensates would likely be associated with high divalent and organic cation concentrations on the basis of our findings with riboswitches (Poudyal et al in press) and Gquadruplexes (Williams et al 2021). Nonetheless, proteins play critical roles in condensate formation and properties.…”

“…Removal of salt stress (Tack et al 2020) would relieve conventional RNA-RNA base pairing interactions, which are favored by high salt, which might also dissolve condensates. On other hand, we have found that aggregates of spermine-promoted Gquadruplexes are eliminated by higher salt (Williams et al 2021), which is the opposite effect, occurring here because the salt competes the spermine away from the RNA. The major takeaway from these studies is that abiotic variables such as temperature and salt can have a multitude of effects on biomolecular condensates such that their dynamics, especially in living systems.…”

“…As such, they could be especially effective in bringing multiple RNA strands together (Khong et al 2017;Zhang et al 2019). Stretches of as few as two G's could allow quadruplex formation, and these can be favored by the presence of polyamines, especially the organic cation spermine (Williams et al 2021). Quartets can form out of bases other than just G as well.…”

Coacervate Formed by an ATP-Binding Aptamer and Its Dynamic Behavior under Nonequilibrium Conditions

“…Therefore, the rigidity and charge density of an aptamer are greater than those of the double-stranded DNA, and liquid–solid phase separation occurs when the aptamer is mixed with PLL under the studied conditions. A similar result was obtained when the dense G-quadruplex was mixed with polycations …”

Nucleobase Clustering Contributes to the Formation and Hollowing of Repeat-Expansion RNA Condensate

“…The stacking/clustering should involve stacking between RNA single-stranded regions [23] or stacking between multiple nucleobases. [24] To map the location-specific RNA structure in the condensed phase, we employed SRM [13a] and collected a hyperspectral stack of Raman images at wavelengths from 650 to 1600 cm -1 for the three types of RNA droplets. After normalizing by the Raman intensity at 1100cm -1 , the intensity differences at other wavelengths become obvious (Figure S5).…”