Target Discovery and Validation Reviews and Protocols

152 Sano and Taira
4. Notes
1. We recommend that the length of ribozyme’s binding arms should be 7 nt each.
A shorter length of the binding arms may reduce the binding ability to a substrate
and longer length may cause nonspecific binding.
2. The PCR purification kit removes enzymes and salts as well as excess primers.
3. To check the quality of plasmids, linearized plasmids also should be performed the
ligation reaction in the absent of the insert DNA. Then, we must confirm that no
or very few transformed colonies appear.
4. Do not incubate E. coli cells for more than 1 h. Since each transformed cell replicates
at different rates, longer incubation may vary the original population of each
cell.
5. In the case of a hammerhead ribozyme, a gap nucleotide described as H (A, C, or U)
does not form a base pairing.
6. Optionally, species restriction may reduce the unrelated sequences. In addition,
ribozymes often target EST (expression sequencing tag), which is the identified
sequences expressing in cells.
Acknowledgment
The authors thank Dr. Renu Wadhwa for a critical reading of the manuscript.
References
1. Eckstein, F. and Lilley, D. M. J., eds. (1996) Nucleic Acids and Molecular
Biology, vol. 10, Springer-Verlag, Berlin
2. Sioud, M., ed. (2004) Ribozymes and siRNA Protocols, Second Edition. Vol. 252,
Methods in Molecular Biology, Humana Press, Totowa NJ.
3. Koseki, S., Tanabe, T., Tani, K., et al. (1999) Factors governing the activity in vivo
of ribozymes transcribed by RNA polymerase III. J. Virol. 73, 1868–1877.
4. Kato,Y., Kuwabara, T., Warashina, M., Toda, H., and Taira, K. (2001) Relationships
between the activities in vitro and in vivo of various kinds of ribozyme and their
intracellular localization in mammalian cells. J. Biol. Chem. 276, 15,378–15,385.
5. Kuwabara, T., Warashina, M., Koseki, S., et al. (2001) Significantly higher
activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear
counterpart: engineered tRNAs with an extended 3’ end can be exported efficiently
and specifically to the cytoplasm in mammalian cells. Nucleic Acids Res.
29, 2780–2788.
6. Suyama, E., Kawasaki, H., Kasaoka, T., and Taira, K. (2003) Identification of genes
responsible for cell migration by a library of randomized ribozymes. Cancer Res.
63, 119–124.
7. Suyama, E., Kawasaki, H., Nakajima, M., and Taira, K. (2003) Identification of
genes involved in cell invasion by using a library of randomized hybrid ribozymes.
Proc. Natl. Acad. Sci. USA 100, 5616–5621.
8. Suyama, E., Wadhwa, R., Kaur, K., et al. (2004) Identification of Metastasisrelated
Genes in a Mouse Model Using a Library of Randomized Ribozymes.
J. Biol. Chem. 279, 38,083–38,086.