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Nucleic Acids Research, 1993, Vol. 21, No. 24 5656-5660
© 1993

RNA

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions

Sinya Sawata1,+, Takashi Shimayama1,§, Makoto Komiyama, Penmetcha K.R. Kumar1, Satoshi Nishikawa1 and Kazunari Taira1,*

Department of Industrial Chemistry, Faculty of Engineering The University of Tokyo, Hongo, Tokyo 113 11 National Institute of Bioscience and Human Technology, Agency of Industrial Science & Technology, MITI Tsukuba Science City 305, Japan

*To whom correspondence should be addressed

Received September 1, 1993. Revised November 1, 1993. Accepted November 1, 1993.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat, values were proven to represent the chemical-cleavage step and not the productdissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2* and Ca2* with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemicalprobing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.

+Institute of Materials Science, University of Tsukuba, Tennoudai, Tsukuba 305

§Hitachi Chemical Co., Wadai, Tsukuba 300-42, Japan


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