| Bioinformatic prediction and databases |
| Several tools predicting quadruplex-forming propensity are based on the fact that runs of Gs are a requirement for G4 formation. A simple, regular motif (G3+N1–7G3+N1–7G3+N1–7G3+) based on DNA G4 folding studies was originally proposed to describe G4-forming sequences (1,2). Several tools are based on this algorithm, including, QuadDB (128), QGRS mapper (129), Quadfinder (130), QuadBase (131), Greglist (132). More recent algorithms and scoring systems take into account both the neighboring sequences (including G4 Hunter (19), cG/cC score (133)) and the observation that G4s are highly polymorphic in vivo (3,134). Tools. QuadDB (1,128), defines G4 folding rules based on strand stoichiometry, number of tetrads, discontinuities in G-tracts, loop length and composition. Quadfinder (130), prediction of G4 sequences with the flexibility of defining variants of the motif. QGRS mapper (129), prediction of G4 sequences with different parameter settings and possibility to look at sequences with few Gs and long loops; includes a scoring parameter. G4P Calculator (134), orthogonal approach focused on the density of sequences likely to lead to G4s. GRSDB2 and GRS_UTRdb (135,136), list G4s in pre-mRNAs and UTRs. QuadBase (131), ortholog analysis for finding conserved G4s. Greglist (132), list of genes that contain G4 motifs in promoters. G4RNA (137), data retrieval on experimentally tested sequences. G4Hunter (19) takes into account G-richness and G-skewness of a given sequence; provides a G4 propensity score. |
| G4 structure |
| Topologies of G4s depends on the glycosidic conformation (syn or anti), the number of molecules of the nucleic acid involved in their formation (intramolecular, bimolecular or tetramolecular) and the relative orientation of the strands (parallel, antiparallel or mixed). G4 formation depends on several parameters: the number of stacking G-quartets, the length and the nucleotide sequence of the loops, the occurrence of bulges within G-tracks, cation availability and concentration, the presence of consecutive cytosine residues in the surrounding sequence. RNA G4s are more thermodynamically stable, compact and less hydrated than DNA G4s. The 2΄-OH group in the ribose exerts conformational constraints on RNA G4s resulting in more intramolecular interactions, anti conformation and parallel topology. Biophysical techniques: Ultraviolet spectroscopy, Circular dichroism, UV melting, NMR spectroscopy, Crystallography. Drawback: the length of the G4 sequence required for these techniques that does not reflect the in cellulo/in vivo global context. |
| In vitro determination |
| The capability of putative quadruplex sequences to fold into G4 could be assessed experimentally with techniques that use the characteristic of G4s to be stabilized by the presence of a cation (K+>Na+>Li+), and to be modified by G4 small-molecule ligands and trans-acting factors. G4 formation is supported by studies on candidate RNA sequences and, more recently, by transcriptome-wide analysis in vitro (31,32) and in vivo (32). G4 RNA candidate approach: Polyacrylamide gel electrophoresis, reverse transcription pausing assay, DMS (dimethyl sulfate)/footprinting analysis, in-line probing (22), the nucleotide resolutive approach SHALiPE (selective 2΄-hydroxyl acylation with lithium ion-based primer extension) (138), and FOLDeR, a method using 7-deaza-G RNA modifications in combination with secondary structure probing allowing to demonstrate the presence of G4s in long RNAs (139). Transcriptome-wide approaches: RNA G4 (rG4) profiling method that couples rG4-mediated reverse transcriptase pausing with sequencing and generates a global in vitro G4 map (31). More recently, widespread formation of RNA G4s in vitro and in vivo was inferred using DMS treatment before profiling of reverse-transcriptase stops (32). |
| G4 small-molecule ligands |
| Several ligands have been shown to be specific for DNA G4s over other types of DNA structures, including porphyrin, acridine, pentacridium, quinacridine, telomestatin, naphtalene diiamide, bisquinolium derivates. Some of these ligands have been shown to also bind RNA G4s with high affinity and specificity. To date, only two molecules have been demonstrated to exhibit selectivity towards RNA G4s over DNA G4s. RNA/DNA G4 binders: i) TMPyP4 exhibits low affinity for both DNA and RNA G4s, poor selectivity for G4s versus duplex DNA and has opposite effects on RNA G4 formation (50,81,83), ii) Bisquinolium derivates (including Phen-DC3, Phen-DC6, 360A or RR82/R110) are potent binders of DNA and RNA G4s and modulate RNA G4-depedent gene expression (16,25,28,52,82). RNA G4 binders: i) CarboxyPDS (carboxy pyridostatin) triggers selectively RNA G4s within a cellular context (23,140), ii) RGB1, a polyaromatic molecule that binds selectively to RNA G4 structures as compared to DNA G4s or other RNA structures (141). |
| In cellulo probing |
| Facing an urgent need for efficient RNA G4 detection in cellulo, molecular probes that can specifically recognize RNA G4 structures in a simple and reliable way have been recently developed. Structure-specific antibody: BG4 binds both DNA and RNA G4s (23,64). Drawback of immunodetection: fixation and permeabilization of the cells could modify G4 formation; no information on the specific sequences involved in G4 folding. RNA G4 fluorescent probes: CyT (fluorogenic cyanine dye) (24), N-TASQ (naphthoTASQ) (142,143); PyroTASQ (pyrene template-assembled synthetic G-quartet) (144); GTFH (G4-triggered fluorogenic hybridization proble), hybrid probe containing a fluorescent light-up moiety specific to a G4 and an oligonucleotide that hybridize with the specific RNA sequence (145); ThT (thioflavin fluorogenic dye) (146). Advantages: direct detection of RNA G4s in untreated cells (no fixation and no permeabilization). |
