Interaction Of Human Telomeric DNA With N-Methyl Mesoporphyrin IX

The remarkable selectivity of N-methyl mesoporphyrin IX (NMM) for G-quadruplexes (GQs) is long known, however its ability to stabilize and bind GQs has not been investigated in detail. Through the use of circular dichroism, UV-visible spectroscopy and fluorescence resonance energy transfer (FRET) melting assay we have shown that NMM stabilizes human telomeric DNA dAG(3)(TTAG(3))(3) (Tel22) and is selective for its parallel conformation to which it binds in 1:1 stoichiometry with a binding constant of similar to 1.0 x 10(5) M-1. NMM does not interact with an antiparallel conformation of Tel22 in sodium buffer and is the second example in the literature, after TOxaPy, of a ligand with an excellent selectivity for a specific GQ structure. NMM\u27s stabilizing ability toward predominantly parallel GQ conformation is universal: it stabilizes a variety of biologically relevant G-rich sequences including telomeres and oncogene promoters. The N-methyl group is integral for selectivity and stabilization, as the unmethylated analogue, mesoporphyrin IX, does not stabilize GQ DNA in FRET melting assays. Finally, NMM induces the isomerization of Tel22 into a structure with increased parallel component in K+ but not in Na+ buffer. The ability of NMM to cause structural rearrangement and efficient stabilization of Tel22 may bear biological significance

Association Of G-Quadruplex Forming Sequences With Human MtDNA Deletion Breakpoints

Background: Mitochondrial DNA (mtDNA) deletions cause disease and accumulate during aging, yet our understanding of the molecular mechanisms underlying their formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are associated with nuclear DNA instability in cancer; recent evidence indicates they can also form in mitochondrial nucleic acids, suggesting that these non-B DNA structures could be associated with mtDNA deletions. Currently, the multiple types of GQ sequences and their association with human mtDNA stability are unknown. Results: Here, we show an association between human mtDNA deletion breakpoint locations (sites where DNA ends rejoin after deletion of a section) and sequences with G-quadruplex forming potential (QFP), and establish the ability of selected sequences to form GQ in vitro. QFP contain four runs of either two or three consecutive guanines (2G and 3G, respectively), and we identified four types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position of each motif set relative to either 5\u27 or 3\u27 unique mtDNA deletion breakpoints, and found that intrastrand QFP sequences, but not ddi QFP sequences, showed significant association with mtDNA deletion breakpoint locations. Moreover, a large proportion of these QFP sequences occur at smaller distances to breakpoints relative to distribution-matched controls. The positive association of 2G QFP sequences persisted when breakpoints were divided into clinical subgroups. We tested in vitro GQ formation of representative mtDNA sequences containing these 2G QFP sequences and detected robust GQ structures by UV–VIS and CD spectroscopy. Notably, the most frequent deletion breakpoints, including those of the common deletion , are bounded by 2G QFP sequence motifs. Conclusions: The potential for GQ to influence mitochondrial genome stability supports a high-priority investigation of these structures and their regulation in normal and pathological mitochondrial biology. These findings emphasize the potential importance of helicases that subsequently resolve GQ to maintain the stability of the mitochondrial genome

Optimized End-Stacking Provides Specificity Of N-Methyl Mesoporphyrin IX For Human Telomeric G-Quadruplex DNA

N-Methyl mesoporphyrin IX (NMM) is exceptionally selective for G-quadruplexes (GQ) relative to duplex DNA and, as such, has found a wide range of applications in biology and chemistry. In addition, NMM is selective for parallel versus antiparallel GQ folds, as was recently demonstrated in our laboratory. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)(3), Tel22, determined in two space groups, P2(1)2(1)2 and P6, at 1.65 and 2.15 angstrom resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5\u27-5\u27 stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient pi-pi stacking. The out-of-plane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. On the basis of the biochemical data, binding of NMM to Te122 does not rely on relatively nonspecific electrostatic interactions, which characterize most canonical GQ ligands, but rather it is hydrophobic in nature. The structural features observed in the NMM-Tel22 complex described here will serve as guidelines for developing new quadruplex ligands that have excellent affinity and precisely defined selectivity