DISSOCIATION OF LONG-CHAIN DUPLEX RNA CAN OCCUR VIA STRAND DISPLACEMENT IN-VITRO - BIOLOGICAL IMPLICATIONS

Hammerhead ribozymes with long antisense flanks (> 50 bases) have been used successfully to inhibit replication of human immunodeficiency vi rus type 1 (HIV-1) in living cells, To explain their increased efficac y 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 dissociatio n of a double-stranded RNA with 56 bp in vitro, Dissociation was obser ved in the presence of single-stranded RNA with sequence complementari ty to one of the duplex strands, A displacement reaction between RNA s ingle strands and the duplex, but not simple dissociation, was strongl y suggested by the concentration dependence of this process, the influ ence of additional non-complementary sequences on the single strand an d by the unusually low Arrhenius activation energy, The strand displac ement reaction was slow in vitro at 37 degrees C and physiological ion ic strength, but was increased to k approximate to 10(3)-10(4)/M/s (si milar to 10(4)-fold) at higher temperatures by cetyltrimethylammonium bromide, This compound is thought to enhance non-sequence-specific ass ociation of nucleic acids in a mechanistically similar way to that in which cellular hnRNP proteins are thought to act, indicating that stra nd displacement can be fast and, more importantly, could be tightly re gulated in vivo.