Abstract
The specificity of delta ribozyme cleavage was investigated using a trans-acting antigenomic delta ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min-1, an apparent Km of 17.9 nM and an apparent second-order rate constant of 1.89 x 10(7) min-1 M-1. The substrate specificity of the delta ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of delta ribozyme cleavage.
Highlights
Like other ribozymes, ␦ ribozymes have a potential application in gene therapy in which an engineered ribozyme is directed to inhibit gene expression by targeting a specific mRNA molecule
It has been demonstrated that a very low concentration (Ͻ0.1 mM) of Ca2ϩ and Mg2ϩ is required for ␦ ribozyme cleavage [3]. ␦ ribozymes have a unique characteristic in their substrate binding, namely that only the 3Ј-portion of the substrate is required for binding to the ribozyme
Several investigations have been performed to address the questions related to the substrate specificity of ␦ ribozymes in both the cis- and transacting forms [2, 5,6,7,8,9,10,11,12], most, if not all, experiments were carried out by randomly changing the base pairing combinations or by introducing mismatches which interfere with the WatsonCrick base pairing between the substrate and the ribozyme in the P1 stem (Fig. 1)
Summary
Two pairs of complementary and overlapping oligonucleotides, representing the entire length of the ribozyme (57 nt), were synthesized and subjected to an annealing process prior to cloning into pUC19. The annealed oligonucleotides were ligated to HindIII and SmaI co-digested pUC19 to give rise to a plasmid harboring the ␦ ribozyme (referred to as p␦RzP1.1). A mutant ribozyme (␦RzP1.2) was constructed by modifying the substrate recognition site of p␦RzP1.1 by ligation of an oligonucleotide containing the altered sequence flanked by restriction endonuclease sites to RsrII/SphI predigested p␦RzP1.1. The sequences of engineered ribozymes were confirmed by DNA sequencing. Plasmids containing wild type and mutant ribozymes were prepared using Qiagen tip-100 (Qiagen Inc.), digested with SmaI, purified by phenol and chloroform extraction, and precipitated for further use as templates for in vitro transcription reactions
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