Comparative analysis of cleavage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymes.
A trans-cleaving asymmetric hammerhead ribozyme directed against an AUC decreases target motif within an RNA specific for human immunodeficiency virus type 1 (HIV-1) was generated. The AUC decreases motif of the target RNA was permutated in order to generate all 12 variants of an NUX decreases 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 was specific for three of the 12 target motifs. The residual sequence context within helices I and III remained unchanged. All 12 combinations resulted in cleavage of the target RNA. Using single-turnover conditions, the detectable cleavage rate constants at 37 degrees C were determined, which varied considerably depending on the NUX decreases motif. The NUC decreases motifs were cleaved more efficiently, with AUC decreases being cleaved best. Comparison with previous studies indicates that the sequence context of the NUX decreases motif plays a major role for the detectable cleavage activity.
Zoumadakis
M
M
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece.
Tabler
M
M
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004279:DNA, Viral; D016337:RNA, Catalytic; D012367:RNA, Viral