Abstract:
:The catalytic function of the hairpin ribozyme has been investigated by modification-interference analysis of both ribozyme and substrate, using ribonucleoside phosphorothioates. Thiophosphate substitutions in two ribozyme domains were examined by using a novel and highly efficient two-piece ribozyme assembled from two independently synthesized oligoribonucleotides. The catalytic proficiency of the two-piece construct (KM = 48 nM, kcat = 2.3 min-1) is nearly identical to that of the one-piece ribozyme. The two-piece ribozyme is essentially unaffected by substitution with thiophosphates 5' to all guanosines, cytidines, and uridines. In contrast, incorporation of multiple adenosine phosphorothioates in the 5' domain of the ribozyme decreases ribozyme activity by a factor of 25. Modification-interference experiments using ribozymes partially substituted with adenosine phosphorothioate suggest that thiophosphates 5' to A7, A9 and A10 interfere with cleavage to a greater extent than substitutions at other sites within the molecule, but the effect is modest. Within the substrate, phosphorothioate substitution does not directly interfere with cleavage, rather, increasing thiophosphate content decreases the stability of the ribozyme-substrate complex. We describe the construction of a hairpin ribozyme containing dinucleotide extensions at its 5' and 3' ends. Full substitution of this molecule with G and C phosphorothioates results in a ribozyme with greatly enhanced stability against cellular ribonucleases without significant loss of catalytic efficiency.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Chowrira BM,Burke JMdoi
10.1093/nar/20.11.2835keywords:
subject
Has Abstractpub_date
1992-06-11 00:00:00pages
2835-40issue
11eissn
0305-1048issn
1362-4962journal_volume
20pub_type
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