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.

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this ‘double-duplex invasion’, a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the α-nitrogen of N -(2-aminoethyl)- d -lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[ N -(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N -(2-aminoethyl)- d -lysine, the invasion successfully occurred even at highly G–C-rich regions [e.g. (G/C) ₇ (A/T) ₃ and (G/C) ₈ (A/T) ₂ ] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N -(2-aminoethyl)- d -lysine, their l -isomers hardly invaded, showing crucial importance of the d -chirality. The promotion of double-duplex invasion by the chiral ( d ) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.