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 (in vitro) using the G4 stabilizer PDS, and G4 chromatin immunoprecipitation sequencing (G4 ChIP-seq) to detect G4s in in situ fixed chromatin (in vivo) using the G4-specific antibody BG4. We recently reported on G4-RNA precipitation and sequencing (G4RP-seq) to assess the in vivo 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.

Epigenomic features of DNA G-quadruplexes and their roles in regulating rice gene transcription.

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice.

How to untie G-quadruplex knots and why?

For over two decades, the prime objective of the chemical biology community studying G-quadruplexes (G4s) has been to use chemicals to interact with and stabilize G4s in cells to obtain mechanistic interpretations. This strategy has been undoubtedly successful, as demonstrated by recent advances. However, these insights have also led to a fundamental rethinking of G4-targeting strategies: due to the prevalence of G4s in the human genome, transcriptome, and ncRNAome (collectively referred to as the G4ome), and their involvement in human diseases, should we continue developing G4-stabilizing ligands or should we invest in designing molecular tools to unfold G4s? Here, we first focus on how, when, and where G4s fold in cells; then, we describe the enzymatic systems that have evolved to counteract G4 folding and how they have been used as tools to manipulate G4s in cells; finally, we present strategies currently being implemented to devise new molecular G4 unwinding agents.