Loop Nucleotide Chemical Shifts as a Tool to Characterize DNA G‐Quadruplexes

DNA G-quadruplexes are known to play myriad functional roles in the cellular context and their structural diversity has diverse applications in various fields of science. Solution-state NMR spectroscopy has been instrumental in characterization of DNA G-quadruplexes across various fields, with recent advancements in the ¹³C/¹H chemical shift-based approach affording rapid and reliable backbone topology identification. In addition to the backbone topology, for a complete 3D structural characterization, the conformational description of the loops is necessary. In this work, it is demonstrated that ¹³C/¹H chemical shifts of propeller, lateral, and diagonal loop conformations provide an avenue towards topology discrimination within a given backbone fold. Nucleotide-based ¹H shifts when evaluated using random forest and k-nearest neighbors machine learning methodologies provide a modest accuracy in predicting loop conformation. The predicted loop information when analyzed in conjunction with the traditional NMR methods allows for a complete characterization of DNA G-quadruplex folds in a convenient and reliable fashion. In addition, analysis of loop resonances highlights variable conformational flexibility motivating detailed characterization of loop dynamics.