Strategy for modeling higher-order G-quadruplex structures recalcitrant to NMR determination.

• NMR is the gold standard for determining the solution structures of G-quadruplexes. • Higher-order G-quadruplexes (xG4s) are often not amenable to characterization by NMR. • We show how NMR can be utilized in an integrative structural biology (ISB) approach to study xG4s. • We illustrate the ISB method and provide resources and best practices for those new to the field. Guanine-rich nucleic acids can form intramolecularly folded four-stranded structures known as G-quadruplexes (G4s). Traditionally, G4 research has focused on short, highly modified DNA or RNA sequences that form well-defined homogeneous compact structures. However, the existence of longer sequences with multiple G4 repeats, from proto-oncogene promoters to telomeres, suggests the potential for more complex higher-order structures with multiple G4 units that might offer selective drug-targeting sites for therapeutic development. These larger structures present significant challenges for structural characterization by traditional high-resolution methods like multi-dimensional NMR and X-ray crystallography due to their molecular complexity. To address this current challenge, we have developed an integrated structural biology (ISB) platform, combining experimental and computational methods to determine self-consistent molecular models of higher-order G4s (xG4s). Here we outline our ISB method using two recent examples from our lab, an extended c-Myc promoter and long human telomere G4 repeats, that highlights the utility and generality of our approach to characterizing biologically relevant xG4s. [ABSTRACT FROM AUTHOR]