Preferential stabilization of G-quadruplex (G4) structures using small-molecule ligands has emerged as an effective approach to develop anticancer drugs. Herein, we report the synthesis of three indole-fused quindoline derivatives with varying lengths of side chains (InqEt1, InqEt2, and InqPr2) as selective ligands for promoter G4 structures. The ligands stabilize the parallel topology of c-MYC and c-KIT1 promoter G4 DNAs over telomeric and duplex DNAs, as evident from the circular dichroism melting and polymerase stop-assay experiments. The lead ligand, InqPr2, downregulates the gene expression of c-MYC and c-KIT in HeLa and HepG2 cells, respectively, leading to apoptotic cell death. Molecular modeling and dynamics studies support the 2:1 binding stoichiometry revealed from the Job plot analysis and show the ligand’s structural features that enable the preferential binding to the parallel G4 structures over other topologies. Our studies show that indole-fused quindoline derivatives can be harnessed as new molecular scaffolds for selective targeting of parallel G4 topologies.

G-quadruplexes (G4s) are four-stranded motifs formed by G-rich nucleic acid sequences. These structures harbor significant biological importance as they are involved in telomere maintenance, transcription, and translation. Owing to their...

Herein we describe results on the pairing properties of synthetic DNA and RNA oligonucleotides that contain nucleotide analogues with a 7-membered sugar ring (oxepane nucleotides). Specifically, we describe the stereoselective synthesis of a set of three oxepane thymine nucleosides (OxT), their conversion to phosphoramidite derivatives, and their use in solid-phase synthesis to yield chimeric OxT-DNA and OxT-RNA strands. The different regioisomeric OxT phosphoramidites allowed for positional variations of the phosphate bridge and assessment of duplex stability when the oxepane nucleotides were incorporated in dsDNA, dsRNA, and DNA–RNA hybrids. Little to no destabilization was observed when two of the three regioisomeric OxT units were incorporated in the DNA strand of DNA–RNA hybrids, a remarkable result considering the dramatically different structure of oxepanes in comparison to 2′-deoxynucleosides. Extensive molecular modeling and dynamics studies further revealed the various structural features responsible for the tolerance of both OxT modifications in DNA–RNA duplexes, such as base–base stacking and sugar–phosphate H-bond interactions. These studies suggest that oxepane nucleotide analogues may find applications in synthetic biology, where synthetic oligonucleotides can be used to create new tools for biotechnology and medicine.

Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven N 2-dG lesions of different steric bulkiness. In the presence of Mg2+ ions, bulky N 2-isobutyl-dG, N 2-benzyl-dG, N 2-methyl(1-naphthyl)-dG, N 2-methyl(9-anthracenyl)-dG, N 2-methyl(1-pyrenyl)-dG, and N 2-methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite N 2-ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn2+ ions. When Mn2+ replaced Mg2+, PrimPol carried out DNA synthesis on all DNA templates with N 2-dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion “skipping” mechanism. The TLS activity of PrimPol opposite N 2-ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1′–C1′ distances for the N 2-isobutyl-dG and N 2-benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.

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