A review of the latest breakthroughs in the structural classification of G4s reveals the multifaceted physiological and pathological functions of G4‐driven condensates, including chromatin organization, assembly of stress granules and paraspeckles, abnormal transcriptional activation, telomere maintenance, neurodegenerative disease‐associated protein aggregation, and viral inclusion body formation.
Abstract
Among non‐classical nucleic acid secondary structures, G‐quadruplexes (G4s) play diverse roles in cellular functions and disease pathogenesis. However, the molecular mechanisms underlying the assembly of endogenous G4s into punctate condensates in cells remain unclear. Biomolecular condensates can arise from weak multivalent intermolecular interactions involving proteins and/or nucleic acids; this phenomenon is frequently linked to liquid–liquid phase separation. Recent research has provided compelling evidence for G4s driving biomolecular condensation. In this review, we first summarize the latest breakthroughs in the structural classification of G4s. In addition to frequently reported intramolecular G4s, intermolecular G4s have also been observed in cellular environments. Next, we discuss the regulatory role of G4s in condensation. Although G4s can independently form condensates, they primarily serve as structural platforms that facilitate condensate formation and regulate their phase transitions. Ultimately, this review reveals the multifaceted physiological and pathological functions of G4‐driven condensates, including chromatin organization, assembly of stress granules and paraspeckles, abnormal transcriptional activation, telomere maintenance, neurodegenerative disease‐associated protein aggregation, and viral inclusion body formation.
G-quadruplexes (G4s) are essential regulatory structures whose biological functions are inseparable from their potential to destabilize the genome. They play critical roles in transcription, replication and chromatin architecture, yet they also contribute to the genomic instability that fuels cancer. This dual role is an inherent consequence of where G4s form in the genome. G4s are enriched at highly active regulatory regions, including promoters, replication origins and topologically associated domain boundaries, where their controlled formation and resolution by helicases and topoisomerases support normal genome function. When this control is lost, the same features that make G4s functional become harmful, leading to R-loop and G-loop accumulation, replication fork stalling and increased conflicts between transcription and replication. Topoisomerase activity, which normally relieves supercoiling stress, can instead generate the DNA double-strand breaks that characterize cancer genomes. Persistent G4 structures also promote micronuclei formation and cytoplasmic DNA accumulation, activating the cGAS-STING innate immune signaling pathway. Here, we discuss these mechanisms and present pan-cancer genomic analyses showing that these processes operate broadly across human tumors. Therapeutically targeting G4s requires balancing their essential regulatory roles with their pathological effects. Understanding this tension is therefore essential for exploiting G4s as therapeutic targets across cancer types.
Anna R. Bartosik, Magda Bujnowska, D. B. Stovall et al.· Critical reviews in biochemi...· 0 citations
G-quadruplex (G4) structures play key physiological roles and serve as functional building blocks in DNA/RNA-based aptamer biosensors. Their function critically depends on their structural stability and nonspecific interactions in cells. However, the behavior of exogenously delivered G4s in living cells remains poorly understood. Here, using 19F NMR, we monitored three representative G4 sequences, HT, hVEGFP, and TBA, in intact Xenopus laevis oocytes, HeLa cells, and derived lysates. We found that G4 structural stability, interactions, and NMR signal detectability are cell-type-dependent. In oocytes, all three G4s maintain detectable folded signals, with TBA showing slight time-dependent degradation. In HeLa cells, folded signals are absent for all three G4s due to intracellular interactions. HT and TBA undergo degradation in the cytoplasm rather than in the nucleus, whereas hVEGFP uniquely remains protected from degradation owing to both its higher thermal stability and protective interactions. These findings highlight the non-negligible effect of the cellular environment and the necessity of studying G4s in their native cellular context. Furthermore, we demonstrated that cell lysates can serve as a practical platform for reflecting G4 stability in intact cells. Collectively, this work deepens our understanding of G4 behavior in cells and provides a basis for improvement of G4-based agents.
Reversible associations of RNAs among themselves, or with RNA binding proteins through the process of phase separation, are found to be important in many different cellular contexts, including cellular responses to stress, gene regulation, development, and disease. Short RNA repeat sequences, which are mostly linked with many repeat expansion neurodegenerative diseases, are found to undergo phase separation and yield protein-free biomolecular condensates in vitro and in cells. However, the physicochemical principles governing phase separation of RNAs considering both sequence and structural aspects, especially for short RNAs, remain elusive. It is intriguing, as well as challenging, to characterize the RNA phase behavior at a submolecular resolution. Based on atomistic enhanced sampling simulations, here we report potential dynamic structural effects in the mutual association properties of a tetra loop containing 14-mer hairpin RNA. We show that the folded and unfolded conformations of the hairpin fragment lead to different energetic barriers for the formation of an associated pair. Unfolded conformation leads to the formation of gel like energetically stable associated phases spontaneously; the folded hairpin motif is found to yield droplet like associated phases accompanying by cations in solution. Overall, our findings illustrate that dynamic association/dissociation is energetically more favorable for folded RNA hairpins, and the presence of additional salt in solution assists the formation of dense droplet like associated phases, while ionic concentration plays a critical role in the formation and stability of the droplet like associated phase. These observations open avenues for exploring structure-based phase separation mechanisms of RNAs in the context of RNA mediated functional biomolecular condensate formation within cells and designing RNA based novel biomaterials.
M. Mondal, Maodong Li, Y. Gao· Journal of Chemical Physics· 0 citations
Supramolecular assemblies regulated by small biomolecules, such as nucleotides, provide key insights into biological organization, particularly in protein aggregation, which is linked to neurodegenerative diseases. While the role of nucleotides as a stabilizer and biological hydrotrope is well-established, their interaction with biological assembly is inherently paradoxical. Furthermore, the interaction of nucleotides with the transient intermediates of self-assembly of proteins and peptides remains largely unexplored. Here, l-phenylalanine (Phe) is employed as a minimal model to investigate nucleotide-mediated regulation in both native and metal-stabilized assemblies. In native Phe assemblies, ATP and ADP exhibit a concentration-dependent disruption of fibrillar morphology, whereas AMP offers a minimal influence. In contrast, within cation-mediated metastable assemblies, nucleotides actively redirect the assembly pathways. Phosphate multiplicity dictates structural outcomes: ATP and ADP stabilize compact spherical-like architectures, while AMP promotes fibrillar aggregates. Mechanistic analysis, supported by imaging techniques and zeta potential, reveals synergistic contributions from multivalent phosphate interaction and nucleoside-mediated interactions.
Suman Choudhury, Priyanka Nath, Huma Tabassum et al.· Journal of Physical Chemistr...· 0 citations
This review discusses how features of protein sequence, such as amino acid composition, interaction motifs, and post-translational modifications, influence condensate aging and highlights how interactions with RNA and lipid membranes modulate condensate behavior by altering interaction networks and interfacial properties.
Rebecca J. Thrush, Francesco A. Aprile· Current Opinion in Structura...· 0 citations
G-quadruplexes are structurally diverse nucleic acid motifs with important biological properties as well as emerging applications in biotechnology and therapeutics. Here, we investigate whether a double-headed bis-guanine (G) nucleotide, GG-a substantial deviation from canonical nucleic acid architecture-can be accommodated within G-quadruplex structures and how its effects depend on sequence context and topology. Using a combination of circular dichroism (CD) spectroscopy, thermal denaturation analysis, UV thermal differential spectroscopy (TDS), fluorescence light-up assays, polyacrylamide gel electrophoresis (PAGE) analysis, and molecular dynamics (MD) simulations, we show that GG can substitute two consecutive guanosines in G-quadruplex-forming oligonucleotides (ONs) and directly participate in G-tetrad formation, but with strong sequence- and position-dependent consequences. GG incorporation is best tolerated in parallel G-quadruplexes and at G-tetrad steps with low native torsional twist, where local unwinding can be accommodated. In a tetramolecular TG4T system, GG positioned near the 3'-end of the G-stack preserved parallel G-quadruplex topology while providing significant thermal stabilization (+21°C). In the antiparallel thrombin-binding aptamer (TBA), a single GG incorporation yielded exceptional stabilization (+34°C) but was accompanied by altered topological signatures. Together, the results presented herein establish GG as a powerful but context-dependent G-quadruplex stabilizer and define design principles for its use in engineered G-quadruplexes and aptamer development.
Krista Urup, Peter Reinholdt, Kasper M. Beck et al.· Chemistry· 0 citations
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