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.

G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G 2 and three G 3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg 2+ . A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy-bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes.G -quadruplexes are four-stranded structures formed in guanine-rich nucleic acids (1-3). Canonical G-quadruplexes are composed of four tracts of consecutive guanines connected by three loops. The guanines in the guanine tracts (G tracts) are packed in a core unit (Fig. 1A) of a stack of multiple G-quartet layers, each with four guanine bases connected by eight Hoogsteen hydrogen bonds (Fig. 1B). G-quadruplex-forming sequences are not randomly distributed in the mammalian genomes but concentrated at physiologically relevant positions (4): for instance, promoters, telomeres, and immuno-globulin switch regions. These facts suggest G-quadruplex structures have implications in physiological processes. Indeed, experimental investigations have demonstrated the physiological function of G-quadruplexes in many aspects (5-8).Studies on G-quadruplexes have mostly focused on sequences described by a consensus of G ≥3 (N 1-7 G ≥3 ) ≥3 , which can potentially form G-quadruplexes of three or more G-quartet layers with three loops of one to seven nucleotides (Fig. 1A) (9, 10). In recent years, the definition describing the capability of G-quadruplex formation has been broadened. Sequences with a loop up to 11 or 15 nucleotides were found capable of forming stable G-quadruplexes when the other two loops are sufficiently short (11, 12). The continuity of guanines in G tracts was also relaxed by the finding of G-quadruplexes with broken (13, 14) or bulged (15, 16) G tracts. Besides these intramolecular G-quadruplexes in single-stranded DNA (ssDNA), ...