H. Peacock et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5002–5005
5005
Figure 5. A portion of the crystal structure (left) and sequence (right) of the malachite green aptamer bound to tetramethylrosamine (in red). Highlighted in yellow are the C2
positions of the two adenosines (A1 and A2) within the three-nucleotide bulge directly adjacent to the ligand intercalation site (blue box). PDB code: 1F1T.
may be contributing to the yield in these cases. This may be due to
increased reactivity of HTPs 2 and 3, in which the bromoacetamide
is located adjacent to an alpha amino acid (Val or Ser, respectively),
rather than to the (4-aminophenyl)methyl moiety.
Supplementary data
Supplementary data associated with this article can be found, in
RNA2 gave conjugate yields similar to those observed for RNA1
with each of the HTPs tested suggesting a similar placement of
reactive thiol in the complexes involving RNAs 1 and 2 (Fig. 4).
As described above, no high-resolution structure of the HTP-bind-
ing aptamer is available to evaluate the positions of the modified
adenosines. However, an RNA aptamer that binds the dye mala-
chite green is similar in sequence to the HTP aptamer used in this
study. Importantly, a crystal structure22 of this aptamer is available
in which a ligand (tetramethylrosamine, an analog of malachite
green) is intercalated into a 50-CpG-30 step adjacent to an AAU loop.
In this structure, the C2 positions of the loop adenosines are within
three angstroms of each other (Fig. 5). The minimal difference in
the reactions of RNAs 1 and 2 observed in this study is consistent
with positioning of the loop adenosines similar to those found in
the AAU loop of the malachite green aptamer.
In summary, we have shown that the complex between an RNA-
binding helix threading peptide and its RNA target can be stabi-
lized by a covalent bond formed via reaction of thiol-containing
RNA and bromoacetamide-HTP. The reaction was inhibited by a
DNA strand that is Watson–Crick complementary to the RNA target
confirming its dependence on the high affinity HTP binding site
found in the RNA alone. These results are important for future
structural studies of HTP–RNA complexes and methods for the dis-
covery of new high affinity HTP analogs via covalent tethering
strategies.
References and notes
1. Cohen, A. S.; Dubikovskaya, E. A.; Rush, J. S.; Bertozzi, C. R. J. Am. Chem. Soc.
2010, 132, 8563.
2. Speers, A. E.; Cravatt, B. F. J. Am. Chem. Soc. 2005, 127, 10018.
3. Aweda, T. A.; Beck, H. E.; Wu, A. M.; Wei, L. H.; Weber, W. A.; Meares, C. F.
Bioconjugate Chem. 2010, 21, 784.
4. Verdine, G. L.; Norman, D. P. G. Ann. Rev. Biochem. 2003, 72, 337.
5. Yang, C. G.; Yi, C.; Duguid, E. M.; Sullivan, C. T.; Jian, X.; Rice, P. A.; He, C. Nature
2008, 452, 961.
6. Yang, W. J.; Fucini, R. V.; Fahr, B. T.; Randal, M.; Lind, K. E.; Lam, M. B.; Lu, W. L.;
Lu, Y. F.; Cary, D. R.; Romanowski, M. J.; Colussi, D.; Pietrak, B.; Allison, T. J.;
Munshi, S. K.; Penny, D. M.; Pham, P.; Sun, J.; Thomas, A. E.; Wilkinson, J. M.;
Jacobs, J. W.; McDowell, R. S.; Ballinger, M. D. Biochemistry 2009, 48, 4488.
7. Baker, B. R.; Lee, W. W.; Tong, E.; Ross, L. O. J. Am. Chem. Soc. 1961, 83, 3713.
8. Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Science 2005, 308, 1318.
9. Butlin, N. G.; Meares, C. F. Acc. Chem. Res. 2006, 39, 780.
10. Ham, Y. W.; Boger, D. L. J. Am. Chem. Soc. 2004, 126, 9194.
11. Wurz, N. R.; Dervan, P. B. Chem. Biol. 2000, 7, 153.
12. Taran, E. A.; Ivanovskaya, M. G.; Gait, M. J.; Shabarova, Z. A. Mol. Biol. 1998, 32,
697.
13. Carlson, C. B.; Vuyisich, M.; Gooch, B. D.; Beal, P. A. Chem. Biol. 2003, 10, 663.
14. Gooch, B. D.; Beal, P. A. J. Am. Chem. Soc. 2004, 126, 10603.
15. Gooch, B. D.; Krishnamurthy, M.; Shadid, M.; Beal, P. A. ChemBioChem 2005, 6,
2247.
16. Krishnamurthy, M.; Gooch, B. D.; Beal, P. A. Org. Lett. 2004, 6, 63.
17. Krishnamurthy, M.; Simon, K.; Orendt, A. M.; Beal, P. A. Angew. Chem., Int. Ed.
2007, 46, 7044.
18. Schirle, N. T.; Goodman, R. A.; Krishnamurthy, M.; Beal, P. A. Org. Biomol. Chem.
2010, 8, 4898.
19. Hamm, M. L.; Piccirilli, J. A. J. Org. Chem. 1997, 62, 3415.
20. Connolly, B.; Rider, P. Nucleic Acids Res. 1985, 13, 4485.
21. Wecker, M.; Smith, D.; Gold, L. RNA 1996, 2, 982.
Acknowledgment
22. Baugh, C.; Grate, D.; Wilson, C. J. Mol. Biol. 2000, 301, 117.
P.A.B. acknowledges the National Institutes of Health for finan-
cial support in the form of grant R01-GM080784.