G-quadruplexes are non-canonical, four-stranded nucleic acid motifs composed of stacked layers of G-quartets that are stabilized by coordinated metal ions. These motifs, which have been implicated in many important biological processes and diseases, can assume a broad array of sizes and topologies, but only a subset thereof is accessible to high-resolution structural techniques. In our laboratory, we apply ultrafast two-dimensional infrared (2D IR) spectroscopy and site-specific isotope labeling to derive structural parameters from signatures of vibrational coupling among stacked guanine bases. We have found that characteristic frequency and lineshape shifts in the guanine carbonyl (C=O) mode, which arise primarily from vibrational coupling and delocalization within parallel-stranded G-quadruplex arrays, can be used to predict the number of ordered G-quartet layers therein. Across a model series of parallel-stranded oligonucleotides with sequences TG_nT, this analysis revealed a limit for ordered stacking and a metal (K⁺) saturation threshold that matches the lengths of guanine tracts observed in genomes and the ionic conditions within cells. In a recent study, we have shown that K⁺ binding to (TG₅T)₄ originates in the central G-quartet at low K⁺ concentrations and propagates to terminal layers at higher K⁺ concentrations, which in turn modulates the sensitivity of fluorescent small-molecule probes for G-quadruplexes. Additionally, we have shown that 2D IR cross-peaks can be used to distinguish between parallel-stranded and antiparallel-stranded structures that contain different mixtures of syn- and anti- guanine base conformations. Using this insight, combined with guanine C=O frequency trends, we accurately predicted the structure of a 30-nucleotide G-quadruplex formed by the Bcl-2 promoter from infrared data alone. We will discuss the advantages of 2D IR spectroscopy compared to established methods, how it can be used to support the design of therapeutic and molecular probes, and opportunities for further improvement via the union of 2D IR, isotope labeling, and calculations.

Sean D Moran Ph.D.

Assistant Professor, Southern Illinois University Carbondale

Appearances