Identifying G-Quadruplex-DNA-Disrupting Small Molecules.

The quest for small molecules that strongly bind to G-quadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV-vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4-disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.

PhpC modulates G-quadruplex-RNA landscapes in human cells.

Stabilizing DNA/RNA G-quadruplexes (G4s) using small molecules (ligands) has proven an efficient strategy to decipher G4 biology. Quite paradoxically, this search has also highlighted the need for finding molecules able to disrupt G4s to tackle G4-associated cellular dysfunctions. We report here on both qualitative and quantitative investigations that validate the G4-RNA-destabilizing properties of the leading compound PhpC in human cells.

The multivalent G-quadruplex (G4)-ligands MultiTASQs allow for versatile click chemistry-based investigations.

Chemical biology hinges on multivalent molecular tools that can specifically interrogate and/or manipulate cellular circuitries from the inside. The success of many of these approaches relies on molecular tools that make it possible to visualize biological targets in cells and then isolate them for identification purposes. To this end, click chemistry has become in just a few years a vital tool in offering practically convenient solutions to address highly complicated biological questions. We report here on two clickable molecular tools, the biomimetic G-quadruplex (G4) ligands MultiTASQ and azMultiTASQ, which benefit from the versatility of two types of bioorthogonal chemistry, CuAAC and SPAAC (the discovery of which was very recently awarded the Nobel Prize of chemistry). These two MultiTASQs are used here to both visualize G4s in and identify G4s from human cells. To this end, we developed click chemo-precipitation of G-quadruplexes (G4-click-CP) and in situ G4 click imaging protocols, which provide unique insights into G4 biology in a straightforward and reliable manner.

Development of a highly optimized procedure for the discovery of RNA G-quadruplexes by combining several strategies.

RNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the self-association of guanine quartets and that are stabilized by monovalent cations (e.g. potassium). rG4s are key elements in several post-transcriptional regulation mechanisms, including both messenger RNA (mRNA) and microRNA processing, mRNA transport and translation, to name but a few examples. Over the past few years, multiple high-throughput approaches have been developed in order to identify rG4s, including bioinformatic prediction, in vitro assays and affinity capture experiments coupled to RNA sequencing. Each individual approach had its limits, and thus yielded only a fraction of the potential rG4 that are further confirmed (i.e., there is a significant level of false positive). This report aims to benefit from the strengths of several existing approaches to identify rG4s with a high potential of being folded in cells. Briefly, rG4s were pulled-down from cell lysates using the biotinylated biomimetic G4 ligand BioTASQ and the sequences thus isolated were then identified by RNA sequencing. Then, a novel bioinformatic pipeline that included DESeq2 to identify rG4 enriched transcripts, MACS2 to identify rG4 peaks, rG4-seq to increase rG4 formation probability and G4RNA Screener to detect putative rG4s was performed. This workflow uncovers new rG4 candidates whose rG4-folding was then confirmed in vitro using an array of established biophysical methods. Clearly, this workflow led to the identification of novel rG4s in a highly specific and reliable manner.

Click-Chemistry-Based Biomimetic Ligands Efficiently Capture G-Quadruplexes In Vitro and Help Localize Them at DNA Damage Sites in Human Cells.

Interrogating G-quadruplex (G4) biology at its deepest roots in human cells relies on the design, synthesis, and use of ever smarter molecular tools. Here, we demonstrate the versatility of biomimetic G4 ligands referred to as TASQ (template assembled synthetic G-quartet) in which a biotin handle was incorporated for G4-focused chemical biology investigations. We have rethought the biotinylated TASQ design to make it readily chemically accessible via an efficient click-chemistry-based strategy. The resulting biotinylated, triazole-assembled TASQ, or BioTriazoTASQ, was thus shown to efficiently isolate both DNA and RNA G4s from solution by affinity purification protocols, for identification purposes. Its versatility was then further demonstrated by optical imaging that provided unique mechanistic insights into the actual strategic relevance of G4-targeting strategies, showing that ligand-stabilized G4 sites colocalize with and, thus, are responsible for DNA damage foci in human cells.

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.

Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.

The G-quadruplex (G4-DNA or G4) is a secondary DNA structure formed by DNA sequences containing multiple runs of guanines. While it is now firmly established that stabilized G4s lead to enhanced genomic instability in cancer cells, whether and how G4s contribute to genomic instability in brain cells is still not clear. We previously showed that, in cultured primary neurons, small-molecule G4 stabilizers promote formation of DNA double-strand breaks (DSBs) and downregulate the Brca1 gene. Here, we determined if G4-dependent Brca1 downregulation is unique to neurons or if the effects in neurons also occur in astrocytes and microglia. We show that primary neurons, astrocytes and microglia basally exhibit different G4 landscapes. Stabilizing G4-DNA with the G4 ligand pyridostatin (PDS) differentially modifies chromatin structure in these cell types. Intriguingly, PDS promotes DNA DSBs in neurons, astrocytes and microglial cells, but fails to downregulate Brca1 in astrocytes and microglia, indicating differences in DNA damage and repair pathways between brain cell types. Taken together, our findings suggest that stabilized G4-DNA contribute to genomic instability in the brain and may represent a novel senescence pathway in brain aging.