1. The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides. The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3′ terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic. Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides. Oligonucleotides comprised of 2′-methoxy-, 2′-fluoro- or 2′-propoxy-nucleosides did not support RNase H1 activity. 2. The Km and Vmax. of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy ‘gapmers’, i.e.; oligonucleotides with 2′-methoxy wings surrounding a deoxynucleotide centre. Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency. The initial rates of cleavage increased as the deoxynucleotide gap size was increased. Multiple deoxynucleotide gaps increased the Vmax. but had little effect on Km. 3. The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied.
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December 1995
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Research Article|
December 01 1995
Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes Available to Purchase
S T Crooke;
S T Crooke
1Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, U.S.A.
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K M Lemonidis;
K M Lemonidis
1Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, U.S.A.
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L Neilson;
L Neilson
1Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, U.S.A.
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R Griffey;
R Griffey
1Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, U.S.A.
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E A Lesnik;
E A Lesnik
1Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, U.S.A.
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B P Monia
B P Monia
1Isis Pharmaceuticals, Inc., Carlsbad, CA 92008, U.S.A.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1995 The Biochemical Society, London
1995
Biochem J (1995) 312 (2): 599–608.
Citation
S T Crooke, K M Lemonidis, L Neilson, R Griffey, E A Lesnik, B P Monia; Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes. Biochem J 1 December 1995; 312 (2): 599–608. doi: https://doi.org/10.1042/bj3120599
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