Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

Abstract

Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron–electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.

Folding of guanine quadruplex molecules–funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies

“…More common is that the folding landscape consists of several (or even numerous) well-separated deep basins and even the final folded state may consist of more than one basin with detectable population. For human telomeric GQ DNA sequences multiple different folds can co-exist at the thermodynamic equilibrium [13,18,19,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. Even when the native state in the equilibrium involves only one detectable basin, other basins can be transiently populated during the folding process.…”

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

“…The P-S analog of T 2 G 4 T 2 was found to be a more potential inhibitor of HIV infection, in vitro [ 175 , 176 ]. Site-directed spin labeling technique was used for investigating GQ conformations by Zhang et al [ 201 ]. Double nitroxide labels were attached to the sulfur atom of a P-S linkage at G3 and G15, the G4 and G15, and the G9 and G15 of the A(htel-21) oligonucleotide.…”

Precise Distance Measurements in DNA G‐Quadruplex Dimers and Sandwich Complexes by Pulsed Dipolar EPR Spectroscopy

“…[41][42][43][44][45][46][47][48][49][50][51] While PDEPR is most commonly used to determine structural constraints in protein systems, its application to distance measurements within nucleic acids has been steadily expanding. [51][52][53][54][55][56][57][58] So far, few reports describe PDEPR-based investigations on G-quadruplexes, [59][60][61] including in-cell spin-labeled G-quadruplexes [62] and quadruplex-metal complex adducts. [63,64] Recently, we incorporated new Cu 2+ -based spin labels into tetramolecular DNA G-quadruplexes of varying Gtetrad count, which allowed us to determine intramolecular distances within the secondary structure with unprecedented accuracy.…”

“…While PDEPR is most commonly used to determine structural constraints in protein systems, its application to distance measurements within nucleic acids has been steadily expanding [51–58] . So far, few reports describe PDEPR‐based investigations on G‐quadruplexes, [59–61] including in‐cell spin‐labeled G‐quadruplexes [62] and quadruplex‐metal complex adducts [63, 64] …”