Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Conditionally active ribozymes: Hammerhead-Mn-miniribozyme cassettes

Active cis-cleaving ribozymes usually undergo complete self-cleavage during their transcription from DNA templates. Use of latent, or conditionally active, ribozymes that can be activated after transcription by the addition of a simple cofactor allows the preparation of cis-cleaving ribozymes in intact form and study of their self-cleavage. Here we described a model cassette comprising hammerhead ribozymes, antisense effector sequences that cause misfolding of the hammerhead ribozymes, and GAAA-miniribozymes having the 7-nt catalytic core, GAAA/UUU. Due to its unique metal cofactor requirements (manganese or cadmium ions, but not magnesium ions) the GAAA-miniribozyme (1-3) is inactive during transcription in the presence of magnesium ions. Addition of manganese ions induces cleavage of the effector sequence. Upon subsequent denaturation in the presence of EDTA, the effector sequence, initially blocking hammerhead ribozyme activity intramolecularly, becomes detached from its complementary hammerhead sequence. Self-cleavage of the intact hammerhead ribozyme can be then activated by addition of magnesium ions and assayed under different conditions.

References and Footnotes
  1. Kazakov, S., Altman, S. (1992) Proc. Natl. Acad. Sci. USA, 89, 7939.
  2. Vogtherr, M., Limmer, S. (1998) FEBS Lett. 433, 301.
  3. Kuo, T-C., Herrin, D.L. (2000) Nucleic Acids Res. 28, 4197.

Brian H. Johnston (1, 2), Tai-Chih Kuo (1), and Sergei A. Kazakov (1)

Somagenics, Inc.(1), 325 E. Middlefield Rd., Mountain View, CA 94043; Department of Pediatrics(2), Stanford University School of Medicine, Stanford, CA 94305
ph: 650-237-0282; Fax: 650-965-0104; Email: bjohnston@somagenics.com