Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy

Nucleic Acids Res. 2017 May 19;45(9):5501-5511. doi: 10.1093/nar/gkx109.

Abstract

Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Survival
  • Fluorine
  • G-Quadruplexes*
  • Humans
  • Macromolecular Substances / metabolism
  • Magnetic Resonance Spectroscopy*
  • Nucleic Acid Conformation
  • Nucleic Acid Denaturation
  • RNA / chemistry*
  • Telomere / chemistry*
  • Temperature
  • Thermodynamics
  • Xenopus laevis

Substances

  • Macromolecular Substances
  • Fluorine
  • RNA