The polarization dependence of 2D IR cross-peaks distinguishes parallel-stranded and antiparallel-stranded DNA G-quadruplexes.

Guanine-rich nucleic acid sequences have a tendency to form four-stranded non-canonical motifs known as G-quadruplexes. These motifs may adopt a wide range of structures characterized by size, strand orientation, guanine base conformation, and fold topology. Using three K+-bound model systems, we show that vibrational coupling between guanine C6 = O and ring modes varies between parallel-stranded and antiparallel-stranded G-quadruplexes, and that such structures can be distinguished by comparison of the polarization dependences of cross-peaks in their two-dimensional infrared (2D IR) spectra. Combined with previously defined vibrational frequency trends, this analysis reveals key features of a 30-nucleotide unimolecular variant of the Bcl-2 proximal promoter that are consistent with its reported structure. This study shows that 2D IR spectroscopy is a convenient method for analyzing G-quadruplex structures that can be applied to complex sequences where traditional high-resolution methods are limited by solubility and disorder.

Infrared Spectroscopy Reveals the Preferred Motif Size and Local Disorder in Parallel Stranded DNA G-Quadruplexes.

Infrared spectroscopy detects the formation of G-quadruplexes in guanine-rich nucleic acid sequences through shifts in the guanine C=O stretch mode. Here, we use ultrafast 2D infrared (IR) spectroscopy and isotope substitution to show that these shifts arise from vibrational delocalization among stacked G-quartets. This provides a direct measure of the sizes of locally ordered motifs in heterogeneous samples with substantial disordered regions. We find that parallel-stranded, potassium-bound DNA G-quadruplexes are limited to five consecutive G-quartets and 3-4 consecutive layers are preferred for longer polyguanine tracts. The resulting potassium-dependent G-quadruplex assembly landscape reflects the polyguanine tract lengths found in genomes, the ionic conditions prevalent in healthy mammalian cells, and the onset of structural disorder in disease states. Our study describes spectral markers that can be used to probe other G-quadruplex structures and provides insight into the fundamental limits of their formation in biological and artificial systems.