G-quadruplexes (G4s) are non-classical DNA or RNA secondary structures that have been first observed decades ago. Over the years, these four-stranded structural motifs have been demonstrated to have significant regulatory roles in diverse biological processes, but challenges remain in detecting them globally and reliably. Compared to DNA G4s (dG4s), the study of RNA G4s (rG4s) has received less attention until recently. In this review, we will summarize the innovative high-throughput methods recently developed to detect rG4s on a transcriptome-wide scale, highlight the many novel and important functions of rG4 being discovered in vivo across the tree of life, and discuss the key biological questions to be addressed in the near future.

RNA G-quadruplexes (rG4s): genomics and biological functions.

Noncoding RNAs are functional transcripts that are not translated into proteins. They represent the largest portion of the human transcriptome and have been shown to regulate gene expression networks in both physiological and pathological cell conditions. Research in this field has made remarkable progress in the comprehension of how aberrations in noncoding RNA drive relevant disease-associated phenotypes; however, the biological role and mechanism of action of several noncoding RNAs still need full understanding. Besides fulfilling its function through sequence-based mechanisms, RNA can form complex secondary and tertiary structures which allow non-canonical interactions with proteins and/or other nucleic acids. In this context, the presence of G-quadruplexes in microRNAs and long noncoding RNAs is increasingly being reported. This evidence suggests a role for RNA G-quadruplexes in controlling microRNA biogenesis and mediating noncoding RNA interaction with biological partners, thus ultimately regulating gene expression. Here, we review the state of the art of G-quadruplexes in the noncoding transcriptome, with their structural and functional characterization. In light of the existence and further possible development of G-quadruplex binders that modulate G-quadruplex conformation and protein interactions, we also discuss the therapeutic potential of G-quadruplexes as targets to interfere with disease-associated noncoding RNAs.

Biological relevance and therapeutic potential of G-quadruplex structures in the human noncoding transcriptome

Progress in the design of G-quadruplex (G4) binding ligands relies on the availability of approaches that assess the binding mode and nature of the interactions between G4 forming sequences and their putative ligands. The experimental approaches used to characterize G4/ligand interactions can be categorized into structure-based methods (circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography), affinity and apparent affinity-based methods (surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS)), and high-throughput methods (fluorescence resonance energy transfer (FRET)-melting, G4-fluorescent intercalator displacement assay (G4-FID), affinity chromatography and microarrays. Each method has unique advantages and drawbacks, which makes it essential to select the ideal strategies for the biological question being addressed. The structural- and affinity and apparent affinity-based methods are in several cases complex and/or time-consuming and can be combined with fast and cheap high-throughput approaches to improve the design and development of new potential G4 ligands. In recent years, the joint use of these techniques permitted the discovery of a huge number of G4 ligands investigated for diagnostic and therapeutic purposes. Overall, this review article highlights in detail the most commonly used approaches to characterize the G4/ligand interactions, as well as the applications and types of information that can be obtained from the use of each technique.

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions.

G-quadruplexes (G4s) are nucleic acid structure motifs that are of significance in chemistry and biology. The function of G4s is often governed by their interaction with G4-binding proteins. Few categories of G4-specific tools have been developed to inhibit G4-protein interactions; however, until now there is no aptamer tool being developed to do so. Herein, we present a novel L-RNA aptamer that can generally bind to D-RNA G-quadruplex (rG4) structure, and interfere with rG4-protein interaction. Using hTERC rG4 as the target for in vitro selection, we report the shortest L-aptamer being developed so far, with only 25 nucleotides. Notably, this new aptamer, L-Apt.4-1c, adopts a stem-loop structure with the loop folding into an rG4 motif with two G-quartet, demonstrates preferential binding toward rG4s over non-G4s and DNA G-quadruplexes (dG4s), and suppresses hTERC rG4-nucleolin interactions. We also show that inhibition of rG4-protein interaction using L-RNA aptamer L-Apt.4-1c is comparable to or better than G4-specific ligands such as carboxypyridostatin and QUMA-1 respectively, highlighting that our approach and findings expand the current G4 toolbox, and open a new avenue for diverse applications.

/pdf/specific-suppression-of-d-rna-g-quadruplex-protein-4638q480zw.pdf

Specific suppression of D-RNA G-quadruplex-protein interaction with an L-RNA aptamer.

Quadruplex structures have been identified in a plethora of organisms where they play important functions in the regulation of molecular processes, and hence have been proposed as therapeutic targets for many diseases. In this paper we report the extensive bioinformatic analysis of the SARS-CoV-2 genome and related viruses using an upgraded version of the open-source algorithm G4-iM Grinder. This version improves the functionality of the software, including an easy way to determine the potential biological features affected by the candidates found. The quadruplex definitions of the algorithm were optimized for SARS-CoV-2. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, 512 potential quadruplex candidates were discovered. These sequences were evaluated by their in vitro formation probability, their position in the viral RNA, their uniqueness and their conservation rates (calculated in over seventeen thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were then compared subsequently to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire viral realm. Sequences found in common with other viral species were further analyzed and characterized. Sequences with high scores unique to the SARS-CoV-2 were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates were then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable therapeutic targets for the SARS-CoV-2.

/pdf/potential-g-quadruplexes-and-i-motifs-in-the-sars-cov-2-4z3t7gn95p.pdf

Potential G-quadruplexes and i-Motifs in the SARS-CoV-2.

Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species.IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

/pdf/rna-g-quadruplex-structures-mediate-gene-regulation-in-22smzag4i4.pdf

Zijing Seng

Papers

RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria.