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), ...

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