Detection of genomic G-quadruplexes in living cells using a small artificial protein

Abstract

G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12–15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

Biomimetic, smart and multivalent ligands for G-quadruplex isolation and bioorthogonal imaging

“…g ., pyridostatin in G4-seq 43 and rG4-seq 44 protocols). 39, 40, 45 In light of the number of potential G4-forming sequences in the human genome and transcriptome, 43, 46-52 and the biological processes they might be involved in, 31, 53-56 the task can seem daunting; however, this offers a virtually unlimited playground for chemical biology investigation, as every new tool and approach could provide alternative and/or complementary information helping to further decipher the complex biology of G4s.…”

G-quadruplexes are transcription factor binding hubs in human chromatin

“…Recently, endogenous expression of a small, engineered G4-binding protein was reported for detection of DNA G4s via ChIP-seq in human cells [ 50 ]. This alternative mapping approach observed G4s to be enriched at promoters, associated with highly expressed genes, and enrichment of certain proteins (FUS, TAF15, RBM14, TARDBP, HNRNPK, PCBP1) at G4 loci.…”

Genome-wide mapping of G-quadruplex structures with CUT&Tag

“…To systematically compare the G4 CUT&Tag with other G4 mapping methods, we performed G4 CUT&Tag in cell lines for which prior genome-wide datasets had been generated. In parallel, we reprocessed the raw data for G4 ChIP-seq (41), G4P-ChIP (42) through the same bioinformatic pipeline as our G4 CUT&Tag data. G4 CUT&Tag demonstrated improved raw data quality compared to both existing methods, yielding tenfold higher fraction of reads under peaks (FriP) than G4 ChIP-seq and by sixfold higher FriP than G4P-ChIP (Figure 1A).…”

“…Methods to precisely map DNA G-quadruplexes in the genome are crucial to study the functions and regulations of G4 in physiological and pathological processes. Genome-wide chromatin-immunoprecipitation and sequencing (ChIP-seq) based mapping methods have been described, using either a G4-specific antibody in an optimized ChIP-Seq protocol (36, 39-41) or an artificial 6.7 kDa G4 probe (G4P) protein for G4 binding and capture (42).…”

“…Moreover, shearing of the crosslinked chromatin by sonication may dissolve or distort G4 structure and also affect antibody recognition. Thus, alternative approaches have been sought to capture G4s under more native conditions: the G4P-ChIP method takes advantage of a peptide from the DHX36 helicase with high affinity and specificity for G4 structures, whereas D1 ChIP used a mononclonal antibody-GFP fusion protein (42, 44). However, both methods rely on expression of the G4 binder in cells, thus requiring the generation of stable cell lines, and it cannot be excluded that the expression of G4 binding proteins affects G4 biology, e.g.…”