Kinetic Selectivity of Complementary Nucleic Acids: bcr-abl-directed Antisense RNA and Ribozymes

Abstract

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versusslow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitroselection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-ablsequence in comparison with either of the wild-type sequences bcror abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abltranscript was specifically hydrolysed out of a mixture containing abland bcrsequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either viahelix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-ablfusion point. Target selectivity was found for a ribozyme that annealed fast via ablsequences and cleaved within the bcrportion of bcr-ablRNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-ablfusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-ablfusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-ablgene expression in Philadelphia chromosome-positive cells.