Nucleic Acids Research, 1994, Vol. 22, No. 3 293-300
© 1994
RNA |
Can hammerhead ribozymes be efficient tools to inactivate gene function?
Institut Jacques Monod du CNRS, Université Paris 7, Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France
*To whom correspondence should be addressed
Received November 5, 1993. Accepted December 20, 1993.
In order to improve hammerhead ribozyme efficiency and specificity, we have analyzed, both in vitro and in vivo, the activity of a series of ribozyme/substrate combinations that have the same target sequence but differ in the length of the ribozyme/substrate duplex or in their structure, i.e., the total length of the RNA. In vitro, we have found that optimal kcat/Km (at 37°C) is obtained when the ribozyme/substrate duplex has a length of 12 bases, which according to the base composition represents a calculated free energy of binding of -16 kcal/mol. We discuss the importance of this value for ribozyme specificity and present strategies that may improve it. Increasing the length of the duplex from 14 to 17 bases (from –19 to –26 kcal/mol) produces a reduced ribozyme activity which is probably due to a slower rate of product dissociation. In addition, inclusion of either the substrate or the ribozyme in a long transcript produces a reduction (10 fold) of the kcat/Km, probably because of a different accessibility of the target sequence. in vivo, the activity of the trans-acting ribozyme was extremely low and detected in only one case: with a ribozyme/substrate duplex length of 13 bases and with both ribozyme and substrate embedded in short RNAs expressed at a very high level. The similarity of the results obtained in vitro and in vivo indicates that it Is possible to use an in vitro system to optimize ribozymes which are to be used in vivo. Satisfactory results were obtained in vivo only with eis-acting ribozymes. Altogether these results suggest that the ribozyme/substrate hybridization step is the limiting step in vivo and therefore it is not clear If ribozymes represent an improvement over antisense RNAs.
+Present addresses: Beckman Research Institute of the City of Hope, Department of Molecular Genetics, 1500 E. Duarte Rd, Duarte, CA 91010
University of California, San Francisco, Department of Biochemistry and Biophysics, CA 94143-0448, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. P. Liu, J. Haasnoot, O. ter Brake, B. Berkhout, and P. Konstantinova Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron Nucleic Acids Res., May 1, 2008; 36(9): 2811 - 2824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Fiola, J.-P. Perreault, and B. Cousineau Gene Targeting in the Gram-Positive Bacterium Lactococcus lactis, Using Various Delta Ribozymes Appl. Envir. Microbiol., January 1, 2006; 72(1): 869 - 879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. G. Ayre, U. Kohler, R. Turgeon, and J. Haseloff Optimization of trans-splicing ribozyme efficiency and specificity by in vivo genetic selection Nucleic Acids Res., December 15, 2002; 30(24): e141 - e141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Zhang, D. W. Ohannesian, E. L. Kreklau, and L. C. Erickson Modulation of 1,3-Bis-(2-chloroethyl)-1-nitrosourea Resistance in Human Tumor Cells Using Hammerhead Ribozymes Designed to Degrade O6-Methylguanine DNA Methyltransferase mRNA J. Pharmacol. Exp. Ther., July 1, 2001; 298(1): 141 - 147. [Abstract] [Full Text]
|
|
|
|
 |
|
 B. G. Ayre, U. Kohler, H. M. Goodman, and J. Haseloff Design of highly specific cytotoxins by using trans-splicing ribozymes PNAS, March 30, 1999; 96(7): 3507 - 3512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Koseki, T. Tanabe, K. Tani, S. Asano, T. Shioda, Y. Nagai, T. Shimada, J. Ohkawa, and K. Taira Factors Governing the Activity In Vivo of Ribozymes Transcribed by RNA Polymerase III J. Virol., March 1, 1999; 73(3): 1868 - 1877. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Ferbeyre, J. Bratty, H. Chen, and R. Cedergren Cell Cycle Arrest Promotes trans-Hammerhead Ribozyme Action in Yeast J. Biol. Chem., August 9, 1996; 271(32): 19318 - 19323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Orita, R. Vinayak, A. Andrus, M. Warashina, A. Chiba, H. Kaniwa, F. Nishikawa, S. Nishikawa, and K. Taira Magnesium-mediated Conversion of an Inactive Form of a Hammerhead Ribozyme to an Active Complex with Its Substrate J. Biol. Chem., April 19, 1996; 271(16): 9447 - 9454. [Abstract] [Full Text] [PDF]
|
|