Nucleotides-1 to-4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage

In the past, the use of delta ribozyme as a therapeutic tool was limited be cause substrate specificity was thought to be determined by only 8 nucleoti des, Recently, we have accumulated evidence suggesting that the substrate s equence upstream of the cleavage site, which is not involved in the binding ,vith the delta ribozyme, appears to be essential in the selection of an ap propriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed s ingle and multiple mutations in positions -1 to -4 and determined the kinet ic parameters of their cleavage using a model antigenomic delta ribozyme, S ome substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently c leaved substrates, The base at each position from -1 to -4 contributes diff erently to the ability of a substrate to be cleaved. An optimal sequence fo r positions -1 to -4 was determined to be -1HRHY-4 (H = U, C, or A). These results shed light on new features that contribute to the substrate require ment of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.