Genome-wide mapping of G-quadruplex DNA: a step-by-step guide to select the most effective method

Abstract

The development of methods that enabled genome-wide mapping of DNA G-quadruplex structures in chromatin has played a critical role in providing evidence to support the formation of these structures in living cells. Over the past decade, a variety of methods aimed at mapping G-quadruplexes have been reported in the literature. In this critical review, we have sought to provide a technical overview on the relative strengths and weaknesses of the genomics approaches currently available, offering step-by-step guidance to assessing experimental needs and selecting the most appropriate method to achieve effective genome-wide mapping of DNA G-quadruplexes.

Fig. 1. Schematic of a G-quadruplex structure (G4). (A) Example of a three-tetrad G-quadruplex structure with schematic of a G-tetrad composed of four co-planar guanines. The O₆ and N₇ of a guanine established hydrogen bonds with N₁ and N₂ of an opposite guanine to form a G-tetrad. The structure is stabilised by a central monovalent cation (M⁺) coordinating O₆ atoms of the four guanines. G4 stabilisation by metal coordination increases following the K⁺ > Na⁺ > >Li⁺. (B) Schematic of different G4 topologies. Top: Intra-molecular G4s, classified according to the orientation of the strands constituting the G4-structure. Bottom: Inter-molecular G4 structures: bimolecular G4 structure on the left and tetramolecular G4 structure on the right. Strands from different molecules composing the G4 structures are in different colours.

Fig. 2. Summary of the techniques currently available to map G4s in the human genome and brief overview of their features. (A) Schematic of G4 ChIP-Seq. The illustration represents a region of the genome forming a G4 structure. The G4-specific antibody, tagged with a 3xFLAG, is targeted by magnetic beads coated with anti-FLAG antibody. (B) Schematic of G4 CUT&Tag. The G4 structure is targeted by a G4-specific antibody tagged with a 3xFLAG; the tag is recognised by an anti-FLAG antibody, targeted by a tertiary IgG antibody, which recruits protein A-Tn5 complex (pA-Tn5) that cleave DNA at the surrounding sites. (C) Schematic of Chem-map. A G4 structure is bound by a biotinylated G4-ligand; the complex is recognised by an anti-biotin antibody, subsequently targeted by a secondary IgG antibody that recruits the pA-Tn5 complex at the site to cleave the DNA surrounding the G4 structure. (D) Schematic of G4access. MNase cleaves DNA in open chromatin regions before G-rich stretches, including sequences folded into G4s.

Fig. 3. Schematic of G4 ChIP-Seq workflow. (1) After cell harvesting, chromatin is cross-linked and extracted from isolated nuclei, then sonicated to obtain DNA fragments of 100–500 bp; (2) cross-linked chromatin fragments are incubated with a G4-specific antibody (BG4 or SG4) to immuno-precipitate G4-containing fragments; (3) anti-FLAG beads are used to isolate fragments containing G4s by binding to the 3xFLAG tag fused BG4 or SG4; (4) washing steps are performed to reduce non-specific interactions with fragments that do not contain G4s; (5) reverse cross-linking is performed to obtain free DNA by heating and enzymatic digestion of proteins with proteinase K; (6) DNA fragments are eluted and barcoded libraries for high-throughput sequencing are prepared.

Fig. 4. Schematic of G4 CUT&Tag workflow. (1) Native cells/nuclei are immobilised to ConA beads; (2) cells/nuclei-ConA beads complexes are incubated with BG4 or SG4 to target genomic G4s; (3) a rabbit anti-FLAG antibody is used to target the 3xFLAG present on BG4 or SG4 antibodies; (4) the samples are incubated with an anti-rabbit antibody to target the rabbit anti-FLAG antibody and recruit pA-Tn5 to the G4 sites; (5) pA-Tn5 digests DNA surrounding the G4 sites and inserts adaptor for preparation of high-throughput sequencing libraries; (6) all the proteins present in the samples are digested by proteinase K treatment, then DNA extracted and libraries for high-throughput sequencing are prepared by PCR amplification with barcoded primers.

Fig. 5. (A) Schematic of Chem-map workflow. (1) Native cells or nuclei are immobilised to ConA beads; (2) cells/nuclei-ConA beads complexes are incubated with biotinylated G4 ligands, which are subsequentially targeted by a rabbit anti-biotin antibody; (3) an anti-rabbit antibody is added to recruit pA-Tn5 to the G4 sites, which tagments DNA surrounding the G4 sites; (4) and (5) DNA is extracted from cells through aqueous phase separation by phenol chloroform, followed by high-salt (NaCl)/ethanol DNA precipitation; (6) DNA is eluted and libraries for high-throughput sequencing are prepared by PCR amplification with barcoded primers. (B) Chemical structures of PDS and PhenDC3 biotinylated ligands used in Chem-map.

Fig. 6. Schematic of G4access workflow. (1) Nuclei are isolated from native cells using a hypertonic buffer; (2) the nuclease MNase digests open chromatin sites in proximity of DNA G-rich stretches; (3) cold temperatures and SDS are used for DNA extraction; (4) the purified DNA fragments are size selected and extracted from agarose gel; (5) and (6) eluted DNA is used to prepare libraries for high-throughput sequencing.

Fig. 7. Current genomic approaches to G4 mapping. Genome-wide G4 mapping techniques are divided according to their main features. These include employment of a probe, type of probe used, the requirement of a fixative to retain chromatin structure, or the possibility of maintaining native conditions.

Silvia Galli

Gem Flint

Lucie Růžičková

Marco Di Antonio