Dramatic effect of single-base mutation on the conformational dynamics of human telomeric G-quadruplex

Abstract

Guanine-rich DNA sequences can form G-quadruplexes. These four-stranded structures are known to form in several genomic regions and to influence certain biological activities. Sometimes, the instability of G-quadruplexes causes the abnormal biological processes. Mutation is a culprit for the destabilization of G-quadruplexes, but the details of mutated G-quadruplexes are poorly understood. In this article, we investigated the conformational dynamics of single-base mutated human telomeric G-quadruplexes in the presence of K+ with single-molecule FRET spectroscopy. We observed that the replacement of single guanine by thymine in a G-track induces various folded structures, i.e. structural polymorphism. Moreover, direct observation of their dynamics revealed that a single-base mutation causes fast unfolding of folded states under physiological conditions. Furthermore, we found that the degree of destabilization varies according to mutation positions. When the central guanine of a G-track is replaced, the G-quadruplexes unfold quickly at any K+ concentrations and temperature. Meanwhile, outer-quartet mutated G-quadruplexes have heterogeneous dynamics at intermediate K+ concentrations and longstanding folded states at high K+ concentrations. Several factors such as base-stacking interaction and K+ coordination are responsible for the different dynamics according to the mutation position.

RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms

“…In one study, the importance of flanking versus the central G-quartet on the stability of DNA or RNA G4 derived from telomeric region was addressed 27 . Biophysical and mutagenesis studies indicated that each G-quartet contributes equally to RNA G4 stability while the central quartet is of greater importance for DNA G4 stability 27 , in agreement with previous observations 28; 29 .…”

Human telomeric G‐quadruplex: thermodynamic and kinetic studies of telomeric quadruplex stability

“…A number of studies that use single-molecule fluorescence resonance energy transfer (smFRET) methods to examine the stability and dynamics of human telomere quadruplexes have appeared [53][54][55][56][57]. Collectively, these studies reveal that in both Na + and K + solution multiple conformational forms coexist.…”

“…A dynamic switching model between unfolded and folded quadruplex conformational forms was proposed that included six distinct states [54]. Single base mutations within the G-tetrad stack were subsequently found to dramatically alter the distribution and dynamics of quadruplex forms [53]. Telomeric G-quadruplex structures with strategic BrG substitutions were studied using smFRET [55].…”

Single-Molecule Studies of Telomeres and Telomerase

“…These techniques include single-molecule Förster resonance energy transfer (smFRET) (48, 57–59, 64, 74, 77, 80, 81, 110, 124), optical tweezers (26, 27, 54, 98), magnetic tweezers (125, 126), combined smFRET–magnetic tweezers (63), atomic force microscopy (AFM) (66), tethered particle motion assays (18, 62), and protein nanopores (4–6, 102, 119). Each approach comes with its own advantages and drawbacks; however, the most widely employed experiments to date have used smFRET or optical/magnetic tweezers.…”

“…More recently, single-molecule experiments have explored the manner in which deviations from canonical telomere DNA sequence influences GQ folding. For example, the impact of sequence mutations (57, 80), change in connecting loop size (87), and DNA damage (6, 33, 119) on the stability of telomere DNA GQ structures have all been measured using single-molecule methods. Collectively, these studies indicate that relatively minor sequence perturbations can exert significant destabilizing effects on telomere GQ folding.…”