C O M M U N I C A T I O N S
on the G-quadruplex stability, we prepared a 4-thiouridine (s4U)-
containing telomeric RNA sequence (Figure S8). Substitution of
oxygen with sulfur in position 4 of U impairs molecular hydrogen
bonding to each other.10 We found that deletion of the U-tetrad
induced a significant decrease in Tm (Figure S8 and Table S1). These
results suggest that the U-tetrad gives an effective enthalpic
contribution to the RNA G-quadruplex stabilization. We noted that
four uridine residues located at the end of the G-quadruple are
favorable for U-tetrad formation than the previously reported RNA
dimer G-quadruplex,11 in which only two uridine residues are
positioned at the ends. The CD melting experiments showed that
the tetramer structure is significantly more stable (with Tm being
20 °C higher) than the dimer G-quadruplex in K+ solution (Figure
S9).
To further characterize the RNA G-quadruplex structure, MALDI-
TOFMS was used to directly observe the G-quadruplex formation.12
We observed the peaks near m/z [7927.2 + nK+] or [7658.9 +
nNa+] in correspondence to the molecular weight of ORN-1 (MW
) 1915.2, m/z ) 4MW + nK+ or 4MW + nNa+) (Figure S10),
suggesting that the RNA G-quadruplex remains stable even in the
gas phase. K+ and Na+ ion adducts are clearly observed for the
G-quadruplex.12b These MALDI-TOFMS data are in excellent
agreement with the results obtained from CD and NMR studies,
indicating a stable telomere RNA G-quadruplex structure. In several
previous studies it has been reported that associated cations can
locate at different positions of the G-quadruplex.9 Different
monovalent cations can therefore alter the electronic states of the
G-quadruplex, resulting in the differences in the 1D NMR spectrum
in Na+ versus K+. Furthermore, this remarkable ∆G implies that
the four extra hydrogen bonds of the U-tetrad are involved,
suggesting that the U-tetrad may be binding an extra K+ or Na+
ion in the central channel of the G-quadruplex. A further indication
of its stability is the observation that imino protons can be detected
in a sample that has been exchanged into D2O several times and
incubated for 24 h in D2O at 40 °C (Figure S11). These imino
peaks are as strong as the nonexchangeable aromatic proton peaks
at ∼8 ppm. We know of no Watson-Crick duplex as stable as
this. In a native PAGE experiment, a single major band was
observed for ORN-1 with increasing K+ and Na+ concentration
(0-200 mM) (Figure S12), indicating a compact G-quadruplex
formation by ORN-1 even at low salt concentration.
for efforts directed toward the design and generation of potent and
selective RNA G-quadruplex-interacting molecules.
Acknowledgment. This work was partially supported by a
Grant-in-Aid for Scientific Research from the Ministry of Education,
Science, Sports, Culture, and Technology of Japan. Support by the
Global COE Program for Chemistry Innovation is also acknowledged.
Supporting Information Available: General method, CD (Figures
S1, S8, S9, Table S1), NMR (Figures S2-S7, S11), MALDI-TOFMS
(Figure S10), PAGE (Figure S12). This material is available free of
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The finding of telomere RNA molecules opens new doors to
better understanding the essential biological role of telomere. There
is a clear need to revisit structural and functional mechanisms of
telomeres accompanying telomere RNA participation. We and two
other groups have focused effort toward the identification of folding
topologies for telomere RNA architectures.11,13 In this study, a key
discovery is our demonstration of a novel U-tetrad that forms the
base of an RNA G-quadruplex and dramatically stabilizes a human
telomeric RNA G-quadruplex structure. The U-tetrad-stabilized
telomeric RNA G-quadruplex structure adds considerably to our
understanding of the diversity of RNA G-quadruplex architectures.
It shows that the structure of base “quartets” is important in RNA
assembly.14 The unique structural feature may provide new targets
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