Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy.
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.
Division of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Hong-Liang
HL
Bao
Division of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Takumi
T
Ishizuka
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahi-dai, Nomi, Ishikawa 923-1292, Japan.
Takashi
T
Sakamoto
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahi-dai, Nomi, Ishikawa 923-1292, Japan.
Kenzo
K
Fujimoto
Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Tamayo
T
Uechi
Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Naoya
N
Kenmochi
Division of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Yan
Y
Xu
D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D005461:Fluorine; D054856:G-Quadruplexes; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000737:chemistry*; D016615:Telomere / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D014982:Xenopus laevis
D046911:Macromolecular Substances; D005461:Fluorine; D012313:RNA