RNA G4, as an integral branch of G4 structure, possesses distinct interactions with ligands compared to the common DNA G4, thus the investigation of RNA G4/ligand interactions might be considered as a fresh breakthrough to improve the biosensing performance of G4/ligand system. In this study, we comparatively explored the structural and functional mechanisms of RNA G4 and DNA G4 in the interaction with ligands, hemin and thioflavin T (ThT), utilizing the classical PS2.M sequence as a model. We found that although the catalytic performance of RNA G4/hemin system was lower than DNA G4/hemin, RNA G4/ThT fluorescence system exhibited a significant improvement (2∼3-fold) compared to DNA G4/ThT, and adenine modification could further enhance the signaling. Further, by exploring the interaction between RNA G4 and ThT, we deemed that RNA G4 and ThT were stacked in a bimolecular mode compared to single-molecule binding of DNA G4/ThT, thus more strongly limiting the structural spin in ThT excited state. Further, RNA G4/ThT displayed higher environmental tolerance and lower ion dependence than DNA G4/ThT. Finally, we employed RNA G4/ThT as a highly sensitive label-free fluorescent signal output system for in situ imaging of isoforms BCR-ABL e13a2 and e14a2. Overall, this study successfully screened a high-performance RNA G4 biosensing system through systematic RNA G4/ligands interaction studies, which was expected to provide a promising reference for subsequent G4/ligand research.

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ∼100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U\'s RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5\'-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.

NRAS mutation is the second most common oncogenic factor in cutaneous melanoma. Inhibiting NRAS translation by stabilizing the G-quadruplex (G4) structure with small molecules seems to be a potential strategy for cancer therapy due to the NRAS protein\'s lack of a druggable pocket. To enhance the effects of previously reported G4 stabilizers quindoline derivatives, we designed and synthesized a novel series of quindoline derivatives with fork-shaped side chains by introducing (alkylamino)alkoxy side chains. Panels of experimental results showed that introducing a fork-shaped (alkylamino)alkoxy side chain could enhance the stabilizing abilities of the ligands against NRAS RNA G-quadruplexes and their anti-melanoma activities. One of them, 10b, exhibited good antitumor activity in the NRAS-mutant melanoma xenograft mouse model, showing the therapeutic potential of this kind of compounds.

G-quadruplexes, intricate four-stranded structures composed of G-tetrads formed by four guanine bases, are prevalent in both DNA and RNA. Notably, these structures play pivotal roles in human telomeres, contributing to essential cellular functions. Additionally, the existence of DNA:RNA hybrid G-quadruplexes adds a layer of complexity to their structural diversity. This review provides a comprehensive overview of recent advancements in unraveling the intricacies of DNA and RNA G-quadruplexes within human telomeres. Detailed insights into their structural features are presented, encompassing the latest developments in chemical approaches designed to probe these G-quadruplex structures. Furthermore, this review explores the applications of G-quadruplex structures in targeting human telomeres. Finally, the manuscript outlines the imminent challenges in this evolving field, setting the stage for future investigations.

RNA G-quadruplexes (D-rG4s) are prevalent in the transcriptome and play crucial regulatory roles in various biological processes. Recently, L-RNA aptamers have been reported to recognize functional rG4s with a strong binding affinity and specificity. However, owing to the poor cell penetration capacity of L-RNA aptamers, their biological applications are currently limited. Herein, we rationally design an L-RNA aptamer-peptide conjugate, Tamra_Ahx_R8_L-Apt.4-1c, which can efficiently translocate into the cytosol and target the rG4 of interest. Notably, we demonstrate diverse regulatory roles of Tamra_Ahx_R8_L-Apt.4-1c on rG4 motif present in different regions of mRNAs and further expand the application in different cell lines. Our novel and biocompatible conjugate enhances the cellular uptake of the L-RNA aptamer, and our robust strategy enables non-canonical RNA structures to be targeted by L-RNA aptamers for gene control in cells.

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. The 5\' untranslated region (UTR) (5\' UTR) of the NRAS mRNA contains a G-quadruplex (G4) that regulates translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5\' UTR of the NRAS mRNA. We used a small molecule microarray screen to identify molecules that selectively bind to the NRAS-G4 with submicromolar affinity. One compound inhibits the translation of NRAS in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends and RT-PCR analysis revealed that the predominant NRAS transcript does not possess the G4 structure. Thus, although NRAS transcripts lack a G4 in many cell lines the concept of targeting folded regions within 5\' UTRs to control translation remains a highly attractive strategy.

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ or PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ and PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against viruses like SARS-CoV-2, which spread fast and accumulate mutations quickly.

The development of site-specific, target-selective and biocompatible small molecule ligands as a fluorescent tool for real-time study of cellular functions of RNA G-quadruplexes (G4s), which are associated with human cancers, is of significance in cancer biology. We report a fluorescent ligand that is a cytoplasm-specific and RNA G4-selective fluorescent biosensor in live HeLa cells. The in vitro results show that the ligand is highly selective targeting RNA G4s including VEGF, NRAS, BCL2 and TERRA. These G4s are recognized as human cancer hallmarks. Moreover, intracellular competition studies with BRACO19 and PDS, and the colocalization study with G4-specific antibody (BG4) in HeLa cells may support that the ligand selectively binds to G4s in cellulo. Furthermore, the ligand was demonstrated for the first time in the visualization and monitoring of dynamic resolving process of RNA G4s by the overexpressed RFP-tagged DHX36 helicase in live HeLa cells.

The interaction between proteins and nucleic acids is a core element of life. Many proteins bind nucleic acids via a sequence-specific manner, but there are also many types of proteins that recognize various structural motifs. Researchers have recently found that proteins that can recognize DNA and RNA G-quadruplexes (G4s) are very important for basic cellular processes, particularly in eukaryotes. Some of these proteins are located outside the nucleus and interact with RNA, potentially affecting miRNA functions in intercellular communication, which is facilitated by small extracellular vesicles (sEVs). Imbalances in the production of sEVs are associated with various pathologies and senescence in humans. The distribution of miRNA into sEVs is regulated by two RNA-binding proteins, Alyref and FUS. Both proteins possess G-rich recognition motifs that are compatible with the formation of RNA parallel G4 structures. This lends credence to the new hypothesis that G4-formation in RNAs and their interaction with G4-binding proteins can affect the fate of miRNAs and control their distribution in sEVs that are associated with senescence and aging.

SCA36 is a neurodegenerative disease mainly caused by the abnormal expansion of the GGGCCT repeat sequence in intron 1 of NOP56. The RNA sequences of this gene are expected to form large amounts of G-quadruplexes in the cytoplasm, which may be a potential intervention and detection target for SCA36. Here, we have developed a small-molecular compound named TCB-1, which shows good selectivity to the G-quadruplex structure, and its fluorescence can be enhanced by hundreds of folds. Interestingly, TCB-1 can avoid lysosome capture, evenly disperse in the cytoplasm, and selectively light up the cytoplasmic RNA G-quadruplexes. This property allows TCB-1 to sensitively detect the increased formation of cytoplasmic RNA G-quadruplexes in SCA36 model cells. This work not only provides new ideas for the design of small-molecule compounds targeting RNA G-quadruplexes in living cells, but also intuitively demonstrates the increased formation of RNA G-quadruplexes caused by NOP56 gene mutation, providing a possible tool for the detection of SCA36.