Guanine-rich RNA sequences can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), whose biological roles are poorly understood, and in vivo existence is debated. To profile biologically relevant G4-RNA in the human transcriptome, we report here on G4RP-seq, which combines G4-RNA-specific precipitation (G4RP) with sequencing. This protocol comprises a chemical crosslinking step, followed by affinity capture with the G4-specific small-molecule ligand/probe BioTASQ, and target identification by sequencing, allowing for capturing global snapshots of transiently folded G4-RNAs. We detect widespread G4-RNA targets within the transcriptome, indicative of transient G4 formation in living human cells. Using G4RP-seq, we also demonstrate that G4-stabilizing ligands (BRACO-19 and RHPS4) can change the G4 transcriptomic landscape, most notably in long non-coding RNAs. G4RP-seq thus provides a method for studying the G4-RNA landscape, as well as ways of considering the mechanisms underlying G4-RNA formation, and the activity of G4-stabilizing ligands.

For more than two decades now, the prime objective of the chemical biology community studying G-quadruplexes (G4s) has been to use chemicals to interact with and stabilize G4s (i.e., G4-ligands) in cells to obtain mechanistic interpretations. This strategy has been undoubtedly successful, as demonstrated by the spectacular recent advances. However, these insights have also led to a fundamental rethinking of G4-targeting strategies: in light of the prevalence of G4s in the human genome, transcriptome and ncRNAome (collectively referred to as the G4ome), and particularly their involvement in human diseases (cancers and neuropathologies), should we continue to search for and develop G4-stabilizing ligands or would it not be wise to invest efforts in designing and exploiting molecular tools able to unfold G4s? Perhaps provocative, this question invites us to take a new and fresh look at the wealth of data accumulated on the functional relevance of G4s. In this review, we first focus on how, when and where G4s fold in cells; then, we describe the enzymatic systems the cells have evolved to counteract G4 folding (i.e., G4-helicases) and how they have been used as biomolecular tools to manipulate G4s in cells; finally, we present the strategies currently being implemented by chemical biologists on G4-ligands and G4-helicase to devise new molecular G4-unwinding agents.