7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges.

We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools.

Transcription-facilitating histone chaperons interact with genomic and synthetic G4 structures.

Affinity for G-quadruplex (G4) structures may be a common feature of transcription-facilitating histone chaperons (HCs). This assumption is based on previous unmatched studies of HCs FACT, nucleolin (NCL), BRD3, and ATRX. We verified this assumption and considered its implications for the therapeutic applications of synthetic (exogenous) G4s and the biological significance of genomic G4s. First, we questioned whether exogenous G4s that recognize cell-surface NCL and could trap other HCs in the nucleus are usable as anticancer agents. We performed in vitro binding assays and selected leading multi-targeted G4s. They exhibited minor effects on cell viability. The presumed NCL-regulated intracellular transport of G4s was inefficient or insufficient for tumor-specific G4 delivery. Next, to clarify whether G4s in the human genome could recruit HCs, we compared available HC ChIP-seq data with G4-seq/G4-ChIP-seq data. Several G4s, including the well-known c-Myc quadruplex structure, were found to be colocalized with HC occupancy sites in cancer cell lines. As evidenced by our molecular modeling data, c-Myc G4 might interfere with the HC function of BRD3 but is unlikely to prevent the BRD3-driven assembly of the chromatin remodeling complex. The c-Myc case illustrates the intricate role of genomic G4s in chromatin remodeling, nucleosome remodeling, and transcription.