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.

Various oligonucleotides containing 8-methylguanine (m⁸G) have been synthesized and their structures and thermodynamic properties investigated. Introduction of m⁸G into DNA sequences markedly stabilizes the Z conformation under low salt conditions. The hexamer d(CGC[m⁸G]CG)₂ exhibits a CD spectrum characteristic of the Z conformation under physiological salt conditions. The NOE-restrained refinement unequivocally demonstrated that d(CGC[m⁸G]CG)₂ adopts a Z structure with all guanines in the syn conformation. The refined NMR structure is very similar to the Z form crystal structure of d(CGCGCG)₂, with a root mean square deviation of 0.6 between the two structures. The contribution of m⁸G to the stabilization of Z-DNA has been estimated from the mid-point NaCl concentrations for the B-Z transition of various m⁸G-containing oligomers. The presence of m⁸G in d(CGC[m⁸G]CG)₂ stabilizes the Z conformation by at least ΔG = −0.8 kcal/mol relative to the unmodified hexamer. The Z conformation was further stabilized by increasing the number of m⁸Gs incorporated and destabilized by incorporating syn-A or syn-T, found respectively in the (A,T)-containing alternating and non-alternating pyrimidine-purine sequences. The results suggest that the chemically less reactive m⁸G base is a useful agent for studying molecular interactions of Z-DNA or other DNA structures that incorporate syn-G conformation.