COMPARATIVE-ANALYSIS OF CLEAVAGE RATES AFTER SYSTEMATIC PERMUTATION OF THE NUX-DOWN-ARROW CONSENSUS TARGET MOTIF FOR HAMMERHEAD RIBOZYMES

A trans-cleaving asymmetric hammerhead ribozyme directed against an AU C down arrow target motif within an RNA specific for human immunodefic iency virus type 1 (HIV-1) was generated, The AUC down arrow motif of the target RNA was permutated in order to generate all 12 variants of an NUX down arrow consensus target motif, wherein N = A, C, G or U and X = A, C or U. Four asymmetric hammerhead ribozymes differing in the nucleotide that is complementary to N were generated, of which each wa s specific for three of the 12 target motifs. The residual sequence co ntext within helices I and III remained unchanged. All 12 combinations resulted in cleavage of the target RNA, Using single-turnover conditi ons, the detectable cleavage rate constants at 37 degrees C were deter mined, which varied considerably depending on the NUX down arrow motif . The NUC down arrow motifs were cleaved more efficiently, with AUC do wn arrow being cleaved best. Comparison with previous studies indicate s that the sequence context of the NUX down arrow motif plays a major role for the detectable cleavage activity.