Best method to determine DNA G-quadruplex folding: The ¹H-¹³C HSQC NMR experiment

NMR spectroscopy is the major method for G-quadruplex structure determination under physiologically relevant solution conditions. Unlike duplex B-DNA, in which all nucleotides adopt an anti glycosidic conformation, the core tetrad-guanines in a G-quadruplex can adopt anti or syn glycosidic conformation depending on the folding structure. An experimental method that can clearly and unambiguously determine syn and anti tetrad-Gs in a G-quadruplex is highly desirable and necessary. In the present study, we exploit the advantages of the ¹H-¹³C HSQC experiment to determine tetrad-G's glycosidic conformation and thus folding topology of G-quadruplexes. We use several examples to demonstrate the clear and straightforward determination of the guanine glycosidic conformations and G-quadruplex folding structures. Moreover, ¹H-¹³C HSQC data can readily identify adenine H2 resonances as well as determine unusual syn conformation in loop and flanking sequences, a challenging task by standard 2D NOESY.