Abstract
The G-quadruplex (GQ) is a well-studied non-canonical DNA structure formed by G-rich sequences found at telomeres and gene promoters. Biological studies suggest that GQs may play roles in regulating gene expression, DNA replication, and DNA repair. Small molecule ligands were shown to alter GQ structure and stability and thereby serve as novel therapies, particularly against cancer. In this work, we investigate the interaction of a G-rich sequence, 5'-GGGTTGGGTTGGGTTGGG-3' (T1), with a water-soluble porphyrin, N-methyl mesoporphyrin IX (NMM) via biophysical and X-ray crystallographic studies. UV-vis and fluorescence titrations, as well as a Job plot, revealed a 1:1 binding stoichiometry with an impressively tight binding constant of 30-50 μM-1 and ΔG298 of -10.3 kcal/mol. Eight extended variants of T1 (named T2 -T9) were fully characterized and T7 was identified as a suitable candidate for crystallographic studies. We solved the crystal structures of the T1- and T7-NMM complexes at 2.39 and 2.34 Å resolution, respectively. Both complexes form a 5'-5' dimer of parallel GQs capped by NMM at the 3' G-quartet, supporting the 1:1 binding stoichiometry. Our work provides invaluable details about GQ-ligand binding interactions and informs the design of novel anticancer drugs that selectively recognize specific GQs and modulate their stability for therapeutic purposes.
Highlights
DNA typically exists in vivo in a well-defined, right-handed double helix
We showed that the planar analogue of Nmethyl mesoporphyrin IX (NMM), mesoporphyrin IX, does not bind Tel22 [9]
The DNA sequences fold into parallel GQs in solution both alone and in complex with NMM, as well as when bound to NMM in the crystal structures
Summary
DNA typically exists in vivo in a well-defined, right-handed double helix. It can adopt several other secondary structures, when in its single-stranded form, such as at the end of telomeres and during replication, transcription, and DNA repair [1]. G-quadruplex (GQ) DNA is a well-studied non-canonical DNA structure formed by the π-π stacking of G-quartets. Each G-quartet is formed by a planar arrangement of four guanines connected by cyclic Hoogsteen hydrogen bonding. GQs are further stabilized by cations, notably K+, that bind in the center between each pair of G-quartets [2]. GQs readily form in vitro, with their thermodynamic stability in physiological buffers often rivaling that of double-stranded DNA
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