C O M M U N I C A T I O N S
Table 1. Dissociation Constants (in µM) for Small Molecule-RNA
Interactions
chosen to assess the primary sequence requirements for the binding
of compound B-12.11 Again, this compound binds octaloop IX
(whose sequence is unrelated to octaloop IV) very tightly, with a
Kd ) 150 nM. The combined binding data for compounds A-26
and B-12 are consistent with the notion that these ligands are
selective for the size of the RNA hairpin loop, rather than its precise
primary sequence.
Years of research have resulted in the development of a paradigm
for small molecule-duplex DNA binding based on the primary
sequence of the DNA.12 In contrast, the development of general
methods for small-molecule-RNA binding has been limited.
However, RNA adopts unique secondary structures that might be
targeted with appropriate small molecule “modules” specific for
various sizes of RNA hairpin loops, bulges, or internal loops.
Described herein is a compound that specifically binds to RNA
tetraloops and another that binds to octaloops; both bind with a
high degrees of specificity over other loop sizes and other secondary
structures. These are the first compounds reported to have such
discrimination between RNA hairpin loops of various sizes. This
stands in contrast to common RNA ligands (such as aminoglyco-
sides) that have been shown to bind promiscuously to a variety of
RNA secondary structures.13 In addition, the strength of the small
molecule-RNA interactions described herein (70-320 nM) com-
pares favorably with most other RNA-ligand complexes, which
typically fall in the low micromolar range.14 It is remarkable that
small perturbations in the linker can lead to compounds of
substantial specificity; the structural basis for this discrimination
is under active investigation and will be reported in due course.
Acknowledgment. This work was supported by the National
Institutes of Health (NIHGMS R01-GM68385) and the Office of
Naval Research (N00014-02-1-0390). P.J.H. is a fellow of the
Alfred P. Sloan foundation.
with fluorescein.8 The four RNA loops were evaluated at five or
more concentrations of the 105 ligands to obtain an estimate of
the dissociation constant. As shown in Figure 1, two compounds
showed selectivity for the RNA tetraloop (A-26 and A-31; blue
squares in Figure 1), and five compounds showed octaloop
specificity (C-1, A-5, B-5, A-8, B-12; red squares in Figure 1).
These compounds that showed reasonable selectivity were re-
tested to obtain dose-dependent curves from which Kd values could
be calculated (Table 1). Compound A-26 showed a strong affinity
(Kd ) 0.3 µM) for RNA tetraloop I, which was >10-fold tighter than
its binding to hexaloop II and octaloop IV, and ∼5-fold tighter than
the binding to heptaloop III. Among the octaloop selective ligands,
compound B-12 showed both excellent affinity for octaloop IV (Kd
) 0.31 µM) as well as substantial specificity (>30-fold in all cases).
On the basis of their selectivity profiles, the RNA binding
specificity of compounds A-26 and B-12 were further evaluated.
In the interest of identifying compounds that are truly specific for
the RNA hairpin loop motif, these size-selective ligands were also
tested against both a RNA duplex and a RNA single-base bulge.
Both A-26 and B-12 appear to be selective for hairpin loops; the
dissociation constant for B-12 with duplex RNA V and bulge-
containing RNA VI is greater than 25 µM, while A-26 binds to
the RNA bulge ∼12-fold weaker than it binds to the tetraloop.
The site of ligand binding was determined for A-26 and B-12
using RNase footprinting; these footprinting experiments confirmed
that the compounds bind to the loop region of the hairpin loops
(see Supporting Information for footprints). The binding of these
compounds to RNA hairpin loops was unaffected by high salt
concentrations, or presence of competitor tRNAs9 (see Supporting
Information). In addition, the binding specificity of A-26 and B-12
was independently confirmed using UV-melting experiments (see
Supporting Information). Finally, the importance of the dimeric
nature of the deoxystreptamine was confirmed through the synthesis
and evaluation of the mono-deoxystreptamine compound 36, which
was not a general RNA loop binder (Table 1).
Supporting Information Available: Full experimental protocols,
binding data, RNase I footprints, and characterization data. This material
References
(1) Hopkins, A. L.; Groom, C. R. Nat. ReV. Drug. DiscoVery 2002, 1, 727-730.
(2) For reviews on targeting RNA see: (a) Sucheck, S. J.; Wong, C.-H. Curr.
Opin. Chem. Biol. 2000, 4, 678-686. (b) Hermann, T. Angew. Chem.,
Int. Ed. 2000, 39, 1890-1904. (c) Michael, K.; Tor, Y. Chem. Eur. J.
1998, 4, 2091-2098.
(3) Examples of targeting mRNA with small molecules: (a) Werstuck, G.;
Green, M. R. Science 1998, 282, 296-298. (b) DeNap, J. C.; Thomas, J.
R.; Musk, D. J.; Hergenrother, P. J. J. Am. Chem. Soc. 2004, 126, 15402-
15404.
(4) Liu, X.; Thomas, J. R.; Hergenrother, P. J. J. Am. Chem. Soc. 2004, 126,
9196-9167.
(5) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596-2599.
(6) For other dimeric RNA binding small molecules, see: (a) Sucheck, S. J.;
Wong, A. L.; Koeller, K. M.; Boehr, D. D.; Draker, K.; Sears, P.; Wright,
G. D.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122, 5230-5231. (b) Tok,
J. B.; Huffman, G. R. Bioorg. Med. Chem. Lett. 2000, 10, 1593-1595.
(7) Stable RNA stem loops were designed using mfold (Mathews, D. H.;
Sabrina, J.; Zuker, M.; Turner, D. H. J. Mol. Biol. 1999, 288, 911-940).
The corresponding pentaloop showed multiple structures in mfold and
was not utilized in this study.
(8) Fluorescently end-labeled RNA to determine small molecule-RNA
Kd’s: (a) Llano-Sotelo, B.; Chow, C. S. Bioorg. Med. Chem. Lett. 1999, 9,
213-216. (b) Llano-Sotelo, B.; Azucena, E. F.; Kotra, L. P.; Mobashery,
S.; Chow, C. S. Chem. Biol. 2002, 9, 455-463. (c) Thomas, J. R.; DeNap,
J. C.; Wong, M. L.; Hergenrother, P. J. Biochemistry 2005, 44, 6800-6808.
(9) Luedtke, N. W.; Liu, Q.; Tor, Y. Biochemistry 2003, 42, 11391-11403.
(10) Varani, G. Annu. ReV. Biophys. Biomol. Struct. 1995, 24, 379-404.
(11) Klinck, R.; Westhof, E.; Walker, S.; Afshar, M.; Collier, A.; Aboul-Ela,
F. RNA 2000, 6, 1423-1431.
(12) Dervan, P. B.; Edelson, B. S. Curr. Opin. Struct. Biol. 2003, 13, 284-299.
(13) Verhelst, S. H.; Michiels, P. J.; van der Marel, G. A.; van Boeckel, C.
A.; van Boom, J. H. ChemBioChem 2004, 5, 937-942.
(14) For another high affinity small molecule-RNA interaction see: Evans,
J. M.; Turner, B. A.; Bowen, S.; Ho, A. M.; Sarver, R. W.; Benson, E.;
Parker, C. N. Bioorg. Med. Chem. Lett. 2003, 13, 993-996.
To assess the binding of A-26 to tetraloops of unrelated sequence,
the GNRA (VII) and the UUCG (VIII) tetraloop sequences were
utilized. These tetraloops are thermodynamically stable and widely
observed in vivo.10 In addition, the UUCG loop is present in the
A-site of the 16S ribosomal RNA. Binding assays indicate that A-26
binds these biologically relevant tetraloops VII and VIII with a
strong affinity (70-100 nM, Table 1).
In contrast to tetraloops, RNA hairpin octaloops are much less
common. The RNA octaloop present in the hepatitis C IRES was
JA051685B
9
J. AM. CHEM. SOC. VOL. 127, NO. 36, 2005 12435