G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment.

G-quadruplexes (G4s) have long been implicated in the regulation of chromatin packaging and gene expression. These processes require or are accelerated by the separation of related proteins into liquid condensates on DNA/RNA matrices. While cytoplasmic G4s are acknowledged scaffolds of potentially pathogenic condensates, the possible contribution of G4s to phase transitions in the nucleus has only recently come to light. In this review, we summarize the growing evidence for the G4-dependent assembly of biomolecular condensates at telomeres and transcription initiation sites, as well as nucleoli, speckles, and paraspeckles. The limitations of the underlying assays and the remaining open questions are outlined. We also discuss the molecular basis for the apparent permissive role of G4s in the in vitro condensate assembly based on the interactome data. To highlight the prospects and risks of G4-targeting therapies with respect to the phase transitions, we also touch upon the reported effects of G4-stabilizing small molecules on nuclear biomolecular condensates.

Abnormal intracellular phase transitions in mutant hnRNP A1 may underlie the development of several neurodegenerative diseases. The risk of these diseases increases upon C9Orf72 repeat expansion and the accumulation of the corresponding G-quadruplex (G4)-forming RNA, but the link between this RNA and the disruption of hnRNP A1 homeostasis has not been fully explored so far. Our aim was to clarify the mutual effects of hnRNP A1 and C9Orf72 G4 in vitro. Using various optical methods and atomic force microscopy, we investigated the influence of the G4 on the formation of cross-beta fibrils by the mutant prion-like domain (PLD) of hnRNP A1 and on the co-separation of the non-mutant protein with a typical SR-rich fragment of a splicing factor (SRSF), which normally drives the assembly of nuclear speckles. The G4 was shown to act in a holdase-like manner, i.e., to restrict the fibrillation of the hnRNP A1 PLD, presumably through interactions with the PLD-flanking RGG motif. These interactions resulted in partial unwinding of the G4, suggesting a helicase-like activity of hnRNP A1 RGG. At the same time, the G4 was shown to disrupt hnRNP A1 co-separation with SRSF, suggesting its possible contribution to pathology through interference with splicing regulation.

Peptides are emerging as versatile platforms in medicine, serving as therapeutic agents, diagnostic probes, and drug delivery vehicles. Their physical state—in a form of monomeric cell-penetrating peptides (CPPs), liquid-like coacervates, or solid amyloid fibrils—critically determines their interaction with cell surfaces and subsequent intracellular trafficking pathways. While the transport of CPPs has been extensively studied, the mechanisms governing the cellular uptake of peptide-based coacervates and fibrils are less understood. This review summarizes the current understanding of the intracellular transport mechanisms of all three distinct peptide states and their complexes or conjugates with cargo molecules. We examine a range of pathways, including direct membrane translocation, several endocytosis subtypes, and phagocytosis-like transport. Particular attention is given to unique aspects observed exclusively for CPPs, coacervates, or fibrils. Further verification and detailed characterization of internalization mechanisms are crucial for the rational design of next-generation peptide-based carriers that allow for precise cargo delivery and therapeutic efficacy.

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.