Optimized End-Stacking Provides Specificity of N-Methyl Mesoporphyrin IX for Human Telomeric G-Quadruplex DNA

Abstract

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 Å 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'-5' 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 π-π 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 Tel22 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.

Binding of G-quadruplexes to the N-terminal Recognition Domain of the RNA Helicase Associated with AU-rich Element (RHAU)

“…mesoporphyrin IX, a quadruplex-specific dye that stacks on the G-tetrad faces (43), is significantly reduced relative to free quadruplex. This mode of recognition is novel relative to the few existing high resolution structures of a peptide bound to an RNA duplex-quadruplex junction (44) and a telomeric protein⅐DNA complex (45).…”

An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

“…To gain further insight as to whether Lin28 RNAs contain G4s, we made use of NMM, a dye that binds selectively to G4s (52)(53)(54)(55). When it binds to G4s, NMM is excited at 399 nm and fluoresces at 614 nm (55).…”

A Label-free “Turn-on” Fluorescence Method for Detecting Mercury Ion in Aqueous Solution Based on N-Methyl Mesoporphyrin IX (NMM)/G-quadruplex DNA

“…[35][36][37] Interestingly, unlike triplex, duplex or single-stranded forms of DNA, G-quadruplex DNA can be selectively recognized by N-methyl mesoporphyrin IX (NMM). 38,39 NMM is a commercially available and has pronounced structural selectivity for G-quadruplex DNA. The X-ray crystal structure of a complex between NMM and G-quadruplex DNA has been reported to facilitate the design of the sensors.…”

“…The X-ray crystal structure of a complex between NMM and G-quadruplex DNA has been reported to facilitate the design of the sensors. 38 Commonly, NMM is weakly fluorescence in the free state, but exhibits a dramatic enhancement upon binding to G-quadruplex DNA.…”