Nucleic Acids Research, Vol 24, Issue 22 4395-4400, Copyright © 1996 by Oxford University Press
M Homann, W Nedbal and G Sczakiel
Hammerhead ribozymes with long antisense flanks (>50 bases) have been
used successfully to inhibit replication of human immunodeficiency virus
type 1 (HIV-1) in living cells. To explain their increased efficacy versus
antisense controls or catalytically inactive derivatives, one can consider
dissociation of the ribozyme-product complex to allow a complete catalytic
cycle. In this work we investigated the dissociation of a double-stranded
RNA with 56 bp in vitro. Dissociation was observed in the presence of
single-stranded RNA with sequence complementarity to one of the duplex
strands. A displacement reaction between RNA single strands and the duplex,
but not simple dissociation, was strongly suggested by the concentration
dependence of this process, the influence of additional non- complementary
sequences on the single strand and by the unusually low Arrhenius
activation energy. The strand displacement reaction was slow in vitro at 37
degrees C and physiological ionic strength, but was increased to k
approximately 10(3)-10(4)/M/s (approximately 10(4)-fold) at higher
temperatures by cetyltrimethylammonium bromide. This compound is thought to
enhance non-sequence-specific association of nucleic acids in a
mechanistically similar way to that in which cellular hnRNP proteins are
thought to act, indicating that strand displacement can be fast and, more
importantly, could be tightly regulated in vivo.
ARTICLES
Dissociation of long-chain duplex RNA can occur via strand displacement in vitro: biological implications
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
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