RNA G4 Research Articles

Visualization of Endogenous RNA G-Quadruplex in Individual Genes in Single Cells.

Single-cell RNA imaging enables the detection of endogenous RNA and the analysis of its abundance, localization, and single-cell heterogeneity, which is powerful in the study of RNA biology. G-quadruplex (G4) is a kind of nucleic secondary structure and plays important roles in various biological processes. To reveal the regulating mechanism of monogenic G4s in a specific biological pathway, it is of great important to visualize endogenous RNA G4 in individual genes. Here, we describe Module Assembled Multifunctional Probes Assay (MAMPA) for single-cell visualization of monogenic RNA G4. This method works well in various cell lines and various RNA G4 analytes and has a low detection limit at ten copies per cell.

Tripodal Quinone-Cyanine G-Quadruplex Ligands as Novel Photosensitizers on Photoinduced Cancer Cell Death.

Guanine-quadruplex (G4) selective photosensitizers have huge potential for photodynamic therapy against various diseases correlated with G4 DNA and G4 RNAs; however, the types of photosensitizer skeletons available are limited. Herein, we investigated the ability of our original G4 ligands, tripodal quinone-cyanine dyes (tpQCy(s)), which were developed as fluorescent probes for G4, to act as photosensitizers for cancer-selective apoptosis inducers. The results indicated that the tpQCy skeleton has great potential for developing G4-targeted cancer-selective photosensitizers for photodynamic therapy. Among the two tpQCys, only QCy(BnBT)3, which has greater G4 selectivity, exhibited photoinduced cytotoxicity in HeLa cell growth, suggesting that the direct oxidation of G4 DNA or RNA is crucial for photoinduced cytotoxicity. RNA-seq analysis using a next-generation sequencing technique revealed that apoptosis was clearly induced by photoirradiation after QCy(BnBT)3 treatment.

Visualizing liquid-liquid phase transitions.

Liquid-liquid phase condensation governs a wide range of protein-protein and protein-RNA interactions in vivo and drives the formation of membrane-less compartments such as the nucleolus and stress granules. We have a broad overview of the importance of multivalency and protein disorder in driving liquid-liquid phase transitions. However, the large and complex nature of key proteins and RNA components involved in forming condensates such as stress granules has inhibited a detailed understanding of how condensates form and the structural interactions that take place within them. In this work, we focused on the small human SERF2 protein. We show here that SERF2 contributes to the formation of stress granules. We also show that SERF2 specifically interacts with non-canonical tetrahelical RNA structures called G-quadruplexes, structures which have previously been linked to stress granule formation. The excellent biophysical amenability of both SERF2 and RNA G4 quadruplexes has allowed us to obtain a high-resolution visualization of the multivalent protein-RNA interactions involved in liquid-liquid phase transitions. Our visualization has enabled us to characterize the role that protein disorder plays in these transitions, identify the specific contacts involved, and describe how these interactions impact the structural dynamics of the components involved in liquid-liquid phase transitions, thus enabling a detailed understanding of the structural transitions involved in early stages of ribonucleoprotein condensate formation.

Pre-Defined Stem-Loop Structure Library for the Discovery of L-RNA Aptamers that Target RNA G-Quadruplexes.

L-RNA aptamers have been developed to target G-quadruplexes (G4s) and regulate G4-mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subjected to high failure rate. By analyzing the previously reported G4-binding L-RNA aptamers, we found that the stem-loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4-SLSELEX-Seq platform specifically for G4 targets by introducing a pre-defined stem-loop structure library during SELEX process. Using G4-SLSELEX-Seq, we rapidly identified an L-RNA aptamer, L-Apt1-12 for EBNA1 RNA G4 (rG4) in just three selection rounds. L-Apt1-12 maintained the stem-loop structure initially introduced, and possessed a unique G-triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. Notably, L-Apt1-12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards EBV-positive cancer cells, highlighting its potential for targeted therapy against EBV-associated cancers. Furthermore, we demonstrate the robustness and generality of G4-SLSELEX-Seq by selecting L-RNA aptamers for another two G4 targets-APP rG4 and HCV-1a rG4, also obtaining high-affinity aptamers in three selection rounds. These findings demonstrated G4-SLSELEX-Seq can be a robust and efficient platform for the selection of L-RNA aptamers targeting rG4.

Pre‐Defined Stem‐Loop Structure Library for the Discovery of L‐RNA Aptamers that Target RNA G‐Quadruplexes

L‐RNA aptamers have been developed to target G‐quadruplexes (G4s) and regulate G4‐mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subjected to high failure rate. By analyzing the previously reported G4‐binding L‐RNA aptamers, we found that the stem‐loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4‐SLSELEX‐Seq platform specifically for G4 targets by introducing a pre‐defined stem‐loop structure library during SELEX process. Using G4‐SLSELEX‐Seq, we rapidly identified an L‐RNA aptamer, L‐Apt1‐12 for EBNA1 RNA G4 (rG4) in just three selection rounds. L‐Apt1‐12 maintained the stem‐loop structure initially introduced, and possessed a unique G‐triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. Notably, L‐Apt1‐12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards EBV‐positive cancer cells, highlighting its potential for targeted therapy against EBV‐associated cancers. Furthermore, we demonstrate the robustness and generality of G4‐SLSELEX‐Seq by selecting L‐RNA aptamers for another two G4 targets‐APP rG4 and HCV‐1a rG4, also obtaining high‐affinity aptamers in three selection rounds. These findings demonstrated G4‐SLSELEX‐Seq can be a robust and efficient platform for the selection of L‐RNA aptamers targeting rG4.

Cross-Effects in Folding and Phase Transitions of hnRNP A1 and C9Orf72 RNA G4 In Vitro.

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.

Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity.

Methyltransferase PRC2 (Polycomb Repressive Complex 2) introduces histone H3K27 trimethylation, a repressive chromatin mark, to tune the differential expression of genes. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, notably, RNA. Research on PRC2-associated RNA has mostly focused on the tight-binding G-quadruplex (G4) RNAs, which inhibit PRC2 enzymatic activity in vitro and in cells. Our recent cryo-EM structure provided a molecular mechanism for G4 RNA inactivating PRC2 via dimerization, but it remained unclear how diverse RNAs associate with and regulate PRC2. Here, we show that a single-stranded G-rich RNA and an atypical G4 structure called pUG-fold unexpectedly also mediate near-identical PRC2 dimerization resulting in inhibition of PRC2 methyltransferase activity. The conformational flexibility of arginine-rich loops within subunits EZH2 and AEBP2 of PRC2 can accommodate diverse RNA secondary structures, resulting in protein-RNA and protein-protein interfaces similar to those observed previously with G4 RNA. Furthermore, we address a recent report that failed to detect PRC2-associated RNAs in living cells by demonstrating the insensitivity of PRC2-RNA interaction to photochemical crosslinking. Our results support the significance of RNA-mediated PRC2 regulation by showing that this interaction is not limited to a single RNA secondary structure, consistent with the broad PRC2 transcriptome containing many G-tract RNAs incapable of folding into G4 structures.

IGF2BP1-Mediated Regulation of CCN1 Expression by Specific Binding to G-Quadruplex Structure in Its 3'UTR.

The intricate regulation of gene expression is fundamental to the biological complexity of higher organisms, and is primarily governed by transcriptional and post-transcriptional mechanisms. The 3'-untranslated region (3'UTR) of mRNA is rich in cis-regulatory elements like G-quadruplexes (G4s), and plays a crucial role in post-transcriptional regulation. G4s have emerged as significant gene regulators, impacting mRNA stability, translation, and localization. In this study, we investigate the role of a robust parallel G4 structure situated within the 3'UTR of CCN1 mRNA in post-transcriptional regulation. This G4 structure is proximal to the stop codon of human CCN1, and evolutionarily conserved. We elucidated its interaction with the insulin-like growth factor 2 binding protein 1 (IGF2BP1), a noncanonical RNA N6-methyladenosine (m6A) modification reader, revealing a novel interplay between RNA modifications and G-quadruplex structures. Knockdown experiments and mutagenesis studies demonstrate that IGF2BP1 binds specifically to the G4 structure, modulating CCN1 mRNA stability. Additionally, we unveil the role of IGF2BP1's RNA recognition motifs in G4 recognition, highlighting this enthalpically driven interaction. Our findings offer fresh perspectives on the complex mechanisms of post-transcriptional gene regulation mediated by G4 RNA secondary structures.

A Cyanine Dye for Highly Specific Recognition of Parallel G-Quadruplex Topology and Its Application in Clinical RNA Detection for Cancer Diagnosis.

G-quadruplex (G4), an unconventional nucleic acid structure, shows polymorphism in its topological morphology. The parallel G4 topology is the most prevalent form in organisms and plays a regulatory role in many biological processes. Designing fluorescent probes with high specificity for parallel G4s is important but challenging. Herein, a supramolecular assembly of the anionic cyanine dye SCY-5 is reported, which selectively identifies parallel G4 topology. SCY-5 can clearly distinguish parallel G4s from other G4s and non-G4s, even including hybrid-type G4s with parallel characteristics. The high specificity mechanism of SCY-5 involves a delicate balance between electrostatic repulsion and π-π interaction between SCY-5 and G4s. Using SCY-5, cellular RNA extracted from peripheral venous blood was quantitatively detected, and a remarkable increase in RNA G4 content in cancer patients compared to healthy volunteers was confirmed for the first time. This study provides new insights for designing specific probes for parallel G4 topology and opens a new path for clinical cancer diagnosis using RNA G4 as a biomarker.

Interaction analysis of RNA G-quadruplex with ligands and in situ imaging application

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.

G-Quadruplex mRNAs Silencing with Inducible Ribonuclease Targeting Chimera for Precision Tumor Therapy.

Ribonuclease targeting chimera (RIBOTAC) represents an emerging strategy for targeted therapy. However, RIBOTAC that is selectively activated by bio-orthogonal or cell-specific triggers has not been explored. We developed a strategy of inducible RIBOTAC (iRIBOTAC) that enables on-demand degradation of G-quadruplex (G4) RNAs for precision cancer therapy. iRIBOTAC is designed by coupling an RNA G4 binder with a caged ribonuclease recruiter, which can be decaged by a bio-orthogonal reaction, tumor-specific enzyme, or metabolite. A bivalent G4 binder is engineered by conjugating a near-infrared (NIR) fluorescence G4 ligand to a noncompetitive G4 ligand, conferring fluorescence activation on binding G4s with synergistically enhanced affinity. iRIBOTAC is demonstrated to greatly knockdown G4 RNAs upon activation under bio-orthogonal or cell-specific stimulus, with dysregulation of gene expressions involving cell killing, channel regulator activity, and metabolism as revealed by RNA sequencing. This strategy also shows a crucial effect on cell fate with remarkable biochemical hallmarks of apoptosis. Mice model studies demonstrate that iRIBOTAC allows selective imaging and growth suppression of tumors with bio-orthogonal and tumor-specific controls, highlighting G4 RNA targeting and inducible silencing as a valuable RIBOTAC paradigm for cancer therapy.

Targeting of G-quadruplex DNA with 99mTc(I)/Re(I) Tricarbonyl Complexes Carrying Pyridostatin Derivatives.

The main goal of this work was to elucidate the potential relevance of (radio)metal chelates of 99mTc and Re targeting G-quadruplex structures for the design of new tools for cancer theranostics. 99mTc provides the complexes with the ability to perform single-photon-emission computed tomography imaging studies, while the Re complexes should act as anticancer agents upon interaction with specific G4 DNA or RNA structures present in tumor tissues. Towards this goal, we have developed isostructural 99mTc(I) and Re(I) tricarbonyl complexes anchored by a pyrazolyl-diamine (Pz) chelator carrying a pendant pyridostatin (PDS) fragment as the G4-binding motif. The interaction of the PDF-Pz-Re (8) complex with different G4-forming oligonucleotides was studied by circular dichroism, fluorescence spectroscopy and FRET-melting assays. The results showed that the Re complex retained the ability to bind and stabilize G4-structures from different DNA or RNA sequences, namely those present on the SRC proto-oncogene and telomeric RNA (TERRA sequence). PDF-Pz-Re (8) showed low to moderate cytotoxicity in PC3 and MCF-7 cancer cell lines, as typically observed for G4-binders. Biodistribution studies of the congener PDF-Pz-99mTc (12) in normal mice showed that the complex undergoes a fast blood clearance with a predominant hepatobiliary excretion, pointing also for a high in vitro stability.

In-gel staining methods of G4 DNA and RNA structures.

G-quadruplexes (G4) are functionally important nucleic acid structures, involved in many cellular pathways. They are often dynamically regulated in cells, which makes detecting them in vivo challenging and dependent on sophisticated technical equipment. Therefore, in vitro studies are commonly performed as a first step to confirm a candidate sequence folds into a G4. Several methods have been developed, each with its individual pros and cons. A highly accessible and quick approach, without the need for specialized equipment, is the detection of G4s in native gels using light-up probes. These molecules become fluorescent after specifically binding to G4s. Several different classes have been discovered, emitting light in various colors, and some possess specificity for certain G4 topologies, which makes them highly versatile tools for G4 visualization. Here, we will explore the general procedure using the light-up probe NMM on RNA G4s and discuss advantages and limitations of this method.

Discovery of a minimalistic DIE-based fluorescent probe for detection of parallel G-quadruplex forms

G-quadruplexes (G4s) are considered to be involved in some key biological processes, leading to the development of a large number of G4 fluorescent probes, which offer possibilities to study G4 dynamics as well as their biological roles. However, the structures of G4s show high polymorphism, which can be classified into parallel, hybrid and antiparallel forms, and the probes targeting a certain topology are limited. In this study, we have developed a minimalistic fluorescent probe by exploiting the disaggregation-induced emission (DIE) principle. The further studies demonstrated that this probe exhibited promising selectivity toward parallel DNA and RNA G4 forms in vitro. Moreover, it was found that this probe could be applied to map the RNA G4s that always form into parallel topologies in live cells, which distinguished it from other reported DIE-based probes that often targeted the mitochondrial or nuclear DNA G4s. To the best of our knowledge, this was the first DIE-based fluorescent probe for mapping cellular RNA G4s.

Promoter R-Loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing.

R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) data sets, we found that many splicing factors, including U2AF1 whose recognition of the 3' splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We also revealed that U2AF1 binds directly to R-loops and DNA G4 structures at a low-nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1's interaction with 3' splicing site and leads to augmented distribution of RNA polymerase II (RNAPII) to promoters over gene bodies, thereby modulating cotranscriptional pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interactions of U2AF1 with these nucleic acid structures, and established a biochemical rationale for U2AF1's occupancy in gene promoters. We also unveiled that interaction with R-loops promotes U2AF1's phase separation, and our work suggests that U2AF1 modulates pre-mRNA splicing by regulating RNAPII's partition in transcription initiation versus elongation.

G-quadruplex forming sequences in the genes coding for cytochrome P450 enzymes and their potential roles in drug metabolism

The majority of drugs are metabolized by cytochrome P450 (CYP) enzymes, primarily belonging to the CYP1, CYP2 and CYP3 families. Genetic variations are the main cause of inter-individual differences in drug response, which constitutes a major concern in pharmacotherapy. G-quadruplexes (G4s), are non-canonical DNA and RNA secondary structures formed by guanine-rich sequences. G4s have been implicated in cancer and gene regulation. In this study, we investigated putative G4-forming sequences (PQSs) in the CYP genes. Our findings reveal a high density of PQSs in the full genes of CYP family 2. Moreover, we observe an increased density of PQSs in the promoters of CYP family 1 genes compared to non-CYP450 genes. Importantly, stable PQSs were also identified in all studied CYP genes. Subsequently, we assessed the impact of the most frequently reported genetic mutations in the selected genes and the possible effect of these mutations on G4 formation as well as on the thermodynamic stability of predicted G4s. We found that 4 SNPs overlap G4 sequences and lead to mutated DNA and RNA G4 forming sequences in their context. Notably, the mutation in the CYP2C9 gene, which is associated with impaired (S)-warfarin metabolism in patients, alters a G4 sequence. We then demonstrated that at least 10 of the 13 chosen cytochrome P450 G4 candidates form G-quadruplex structures in vitro, using a combination of spectroscopic methods. In conclusion, our findings indicate the potential role of G-quadruplexes in the regulation of cytochrome genes, and emphasize the importance of G-quadruplexes in drug metabolism.

Peptides Selected by G4-mRNA Display-Seq Enable RNA G-Quadruplex Recognition and Gene Regulation.

G-quadruplexes (G4s) are noncanonical secondary structures that play critical roles in both chemistry and biology. Although several approaches have been developed for G4 targeting, such as chemicals and antibodies, there is currently no general and efficient platform for G4-specific peptides. In this study, we developed a new platform, G4-mRNA display-Seq, for selecting peptides that specifically recognize the G4 target of interest. By using an RNA G4 (rG4) found in human telomerase RNA (hTERC) as the target, we have identified a novel short peptide, namely, peptide 11 (pep11), which displays high affinity and selectivity to hTERC rG4. Furthermore, we designed tandem and cyclic versions of pep11 and found that both modified versions exhibit stronger binding affinity with preferential rG4 selectivity. Notably, we have demonstrated that these peptides can negatively regulate gene expression by targeting rG4. Our results provide a universal platform for the discovery of G4-targeting peptides and demonstrate the ability of these peptides to regulate G4-mediated gene functions.

Single-molecule recognition of Nucleolin and the interactions with DNA/RNA G-quadruplexes via nanopore decoding

DNA/RNA quadruplexes (G4) are significant secondary structures that exist in human genes, playing essential part in transcription and replication. Nucleolin (NCL) is a ubiquitous protein mainly found in the human nucleus. It is frequently overexpressed in cancer cells and displays a remarkable preference for DNA/RNA G4 binding, playing a crucial role in controlling nucleic acid metabolism and chromatin structure. Additionally, NCL is involved in microRNA (miRNA) biogenesis.This work presents the discrimination of DNA/RNA G4 topologies harboring close sequence length with size-matched nanopore devices. The single-molecule identification of NCL under various parameters was implemented, and a low detection limit of 0.5 nM was achieved, NCL could specifically bind with DNA G4 AS1411 and RNA G4 (rG4) from pre-miRNA 92b and pre-miRNA 149, forming stable complexes that demonstrate distinct nanopore translocation properties. The augmentation of the molar ratio of G4 to NCL induces elevated complex formation. Recognition of both NCL and NCL-G4 complex in human serum is realized. Our work established a precise platform for the differentiation of nucleic acid secondary structure with similar conformation, we also proved the binding behaviors between NCL and DNA/RNA G4, as well as the serum interference test, which is valuable for the investigation of the interactions between proteins and nucleic acids as well as for further understanding the mechanism of the NCL-related diseases processes.

Engineering a Red Fluorescent Protein Chromophore for Visualization of RNA G-Quadruplexes.

Synthetic red fluorescent protein (RFP) chromophores have emerged as valuable tools for biological imaging and therapeutic applications, but their application in the visualization of endogenous RNA G-quadruplexes (G4s) in living cells has been rarely reported so far. Here, by integrating the group of the excellent G4 dye ThT, we modulate RFP chromophores to create a novel fluorescent probe DEBIT with red emission. DEBIT selectively recognizes the G4 structure with the advantage of strong binding affinity, high selectivity, and excellent photostability. Using DEBIT as a fluorescent indicator, the real-time monitoring of RNA G4 in biological systems can be achieved. In summary, our work expands the application of synthetic RFP chromophores and provides an essential dye category to the classical G4 probes.

Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs

The search for G-quadruplex (G4)-forming sequences across the genome is motivated by their involvement in key cellular processes and their putative roles in dysregulations underlying human genetic diseases. Sequencing-based methods have been developed to assess the prevalence of DNA G4s genome wide, including G4-seq to detect G4s in purified DNA (invitro) using the G4 stabilizer PDS, and G4 chromatin immunoprecipitation sequencing (G4 ChIP-seq) to detect G4s in in situ fixed chromatin (invivo) using the G4-specific antibody BG4. We recently reported on G4-RNA precipitation and sequencing (G4RP-seq) to assess the invivo prevalence of RNA G4 landscapes transcriptome wide using the small molecule BioTASQ. Here, we apply this technique for mapping DNA G4s in plants (rice) and compare the efficiency of this new technique, G4-DNA precipitation and sequencing, G4DP-seq, to that of BG4-DNA-IP-seq that we developed for mapping of DNA G4s in rice using BG4. By doing so, we compare the G4 capture ability of small-sized ligands (BioTASQ and BioCyTASQ) versus the antibody BG4.