G-quadruplex (NRQ, 5¢-GGGAGGGGCGGGUCUGGG-3¢) in
the presence of aliquots of a stock solution of RR110. The RNA
concentration of the sample was 200 mM in 7% D2O (v/v), buffered
with 10 mM potassium phosphate at pH 7.0. The stock solution
of molecule RR110 was prepared with d6-DMSO (99.9%) at a
concentration of 42 mM. Aliquots of the stocks solution of RR110
were added to the NMR sample in steps to reach molar ratios of
0, 0.25, 0.5, 1, 2, 3, 4 and 5. The final NMR sample contained
2.5% of d6-DMSO. 1H NMR spectra of the RNA G-quadruplex
in the presence of 2.5% DMSO confirmed that DMSO has little
effect to the structure and dynamics of the folded G-quadruplex
(Fig. S3 in the ESI†).
Conclusions
In conclusion, our study demonstrates proof-of-concept for small
molecule-mediated regulation of translation by targeting a natural
RNA G-quadruplex in a 5¢ UTR. While there has been consider-
able attention on genomic DNA G-quadruplexes as potential drug
targets, this current study suggests that RNA G-quadruplexes have
appeal as targets for small molecule interference. Improvement in
the selectivity and potency of the small molecule are now desirable
goals that will be addressed in future studies.
Experimental section
For the hydrogen-deuterium exchange (HDX) experiments,
RNA NMR samples (200 mM, 200 mL) were flash frozen in liquid
nitrogen and lyophilized. Equal volumes (200 mL) of D2O (99.9%)
were then added to resuspend the NMR samples immediately
before data acquisition. Three samples were prepared containing
either no additional material, or 5 mL of the stock solution of
RR110, or 5 mL of d6-DMSO (99.9%). A series of 1D proton
spectra were recorded to follow the kinetics of HDX over 24 h
with a dead-time of ca. 4 min. For each 1D spectrum, 256
transients were recorded and the experimental time, i.e., time
resolution, is 6 min. The resulting spectra were processed and
baseline corrected prior to analysis of the HDX rates. The HDX
kinetics of observed resonances can be generally grouped into
slow, medium and fast phases, as visualized by overlaying the 1D
proton spectra at different time points (Fig. S3 in the ESI†). To
obtain apparent HDX rates of the imino protons of the NRQ
(Fig. S4 and Table S1 in the ESI†), the overall imino proton
Chemical synthesis
Synthesis of RR82 has been reported previously.13 A general
scheme for the synthesis of RR110 is shown in Scheme S1 in the
ESI.† A complete description of the synthesis and characterization
of RR110 is given in the ESI.†
In vitro translation assays
Construction of the plasmids pSKC11, pSKC14 and pSKC17,
which encode for reporter NRAS UTR(+)Q, NRAS UTR(-)Q
and NRAS UTR(+120)Q transcripts, respectively, and conditions
for in vitro transcription have been described previously.10,16 In vitro
translation reactions of the mRNAs in the presence of ligands
were carried out in a cell-free translation system consisting of
extracts from nuclease-treated rabbit reticulocyte lysate (RRL)
(Promega). Typically, 10 mL final volume translation mixtures
were prepared that contain 70% (v/v) RRL, 10 mM amino acid
mixtures minus methionine, 10 mM amino acid mixture minus
leucine, 200 ng RNA and the indicated amount of small molecule
ligand. The RNA was incubated with the small molecule [added
as a 10¥ solution prepared by serial dilution from a 1 mM stock
solution in water (RR82) or water/2.5% DMSO (RR110)] and
the amino acids, in a total volume of 3 mL, for 30 min at room
temperature, prior addition of 7 mL of RRL to start translation.
Translation was carried out at 30 ◦C for 90 min. Translation
efficiency was assessed by measuring firefly luciferase activity using
Luciferase Assay Reagent (Promega) on an Orion II Microplate
Luminometer (Berthold). Typically, 50 mL of luciferase assay
reagent were added to 4.5 mL of in vitro translation mixture. The
luciferase light intensity was measured for 10 s after a delay time
of 2.05 s.
1
signals were integrated (over the range of d H 10.3–11.5 ppm)
and normalized as a function of the exchange time and fitted to a
sum of three exponentially decaying functions: I(t) = Asexp(-kst)
+ Amexp(-kmt) + Afexp(-kft), where As, Am and Af are the initial
amplitudes of the slow, medium and fast phases, respectively, and
ks, km and kf are the corresponding rate constants.
mRNA stability experiments
Translation experiments in the absence or presence (10 mM) of
the small molecule were performed as described above in 80 mL
final volume reaction mixtures with 32P-UTP in vitro transcribed
mRNAs. Aliquots (20 mL) of the translation mixtures were taken
out after 0, 20, 40 and 60 min, quickly frozen in dry-ice and stored
◦
R
at -20 C, before being subjected to Trizolꢀ extraction (500 mL)
◦
and isopropanol precipitation (90 min at -20 C). Samples were
Competition experiments were performed in the same con-
ditions at 10 mM ligand concentration and in the presence
of (i) a 26-mer self-complementary double-stranded DNA (5¢-
CAATCGGATCGAATTCGATCCGATTG-3¢), (ii) an hairpin
RNA (5¢-CUACAGUACAGAUCUGUACUGUAG-3¢), or (iii)
the NRQ NRAS RNA G-quadruplex (5¢- GGGAGGGG-
CGGGUCUGGG-3¢).
run at 70 V for 40–45 min on 2% agarose gels. Gels were dried under
vacuum at 60 ◦C and quantified on an Amersham Biosciences
Typhoon Trio with a Amersham imaging screen.
Acknowledgements
We thank the BBSRC for project funding, Cancer Research
UK for program funding, and the Cambridge Commonwealth
Trust and Trinity College, Cambridge for studentship funding
(S.K.). S-T.D.H. is a recipient of a Netherlands Ramsay Memo-
rial Fellowship and a Human Frontier Long-term Fellowship
(LT0798/2005). We thank the staff and the use of the Biomolecular
NMR Facility, Department of Chemistry, University of Cam-
bridge.
1H NMR experiments
All NMR data were recorded at 298 K using a 700 MHz Bruker
Advance NMR spectrometer, equipped with a TXI cryogenic
probe. The water suppression was achieved by using the jump-
and-return scheme. The NMR titration experiments were carried
out by recording a series of 1D proton NMR spectra of the RNA
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2771–2776 | 2775
©
Bugaut, Anthony
