Construction of a series of several self‐cleaving RNA duplexes using synthetic 21‐mers

Abstract

Two fragments (21-mers) containing consensus sequences for the self-cleavage domain in transcripts of satellite DNA of the newt were chemically synthesized and found to be cleaved in the presence of M$+. The cleaved product contained the 3'-terminal 2',3'-cyclic phosphate. Twenty-five combinations of partially double-stranded 21-mer RNA which contained different bases within the consensus sequences and at the cleavage sites were tested for self-cleavage. It seemed that guanosine 3'-phosphate was not susceptible to transesterification at the cleavage site.

Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

“…The relative cleavage rates of all four studies, standardized to GUC4 = 100%, are summarized in Table 1, which also indicates the neighboring nucleotides 16.3 and 1.1. There are marked differences in the relative cleavage efficiencies regardless of whether short synthetic RNAs were used (9,10) or ribozymes with at least one long antisense flank ( 11) as in this study. This indicates that cleavage motifs cannot easily be categorized into strong and weak motifs.…”

“…motifs, there are three previous studies in which NUC4. and GUXt motifs have been compared in the same sequence context (9)(10)(11). For each set of experiments another catalytic domain had been used.…”

“…The GUCl motif found in nature represents nucleotides 16.2, 16.1 and the unpaired nucleotide 17 according to the numbering system for hammerhead ribozymes by Hertel et al (8). This motif has been converted to AUC4, CUC4, UUC4, GUA4 or GUUt each by Koizumi et al and Ruffner et al (9,10), without loss of activity, even though the cleavage efficiencies varied among the motifs and were generally lower compared with the GUC4 motif. These studies allowed the conclusion that a consensus sequence 'UX4' or 'NUX ' can be described wherein N can be any nucleotide and X any but G. In a similar study, Perriman et al (11) …”

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

“…A consensus secondary structure of approximately 55 nucleotides termed the "hammerhead" domain has been identified (Buzayan et al, 1986b; Forster & Symons, 1987a,b) which can be assembled from 2 or 3 separate RNA molecules. Thus, the self-cleaving reaction has been turned into a multiple turnover reaction, with separate oligonucleotides acting as the "ribozyme" and the "substrate" (Figure 1) (Sampson et al, 1987;Uhlenbeck, 1987;Haseloff & Gerlach, 1988;Koizumi et al, 1988 Koizumi et al, , 1989 Jeffries & Symons, 1989 p3WAACGUC>p; PI-G, GGGAACGUCG; P1-3'p, GGGAACGUC3'-p; P2, GUCGUCGC; P2-C, GUCGUCGCC; P2-p'zCp, GUCGUCGCp"Cp; S-C, GG-G AACGUCGUCGUCGCC; p3*S-C, p3WAACGUCGUCGUCGCC; S-p'zCp, GGGAACGUCGUCGUCGCpWp; S, GGGAACGUC-GUCGUCGC; p32S, p32GGGAACGUCGUCGUCGC; E, ribozyme; HH16, hammerhead cleavage motif comprised of separate ribozyme and substrate oligonucleotides ( (HH 16). Using standardized hammerhead nomenclature (Hertel et al, 1992), the ribozyme (E), comprised of 38 nucleotides, catalyzes thecleavageof a specific phosphodiester bond within its 1 8-nucleotidelong substrate (S-C), generating two 9-nucleotide-long products.…”

Ribozymes Targeted to Stearoyl–ACP Δ9 Desaturase mRNA Produce Heritable Increases of Stearic Acid in Transgenic Maize Leaves

“…Engineered ribozymes have been shown to cleave substrate RNAs in trans (Uhlenbeck, 1987) and have been refined further such that most of the conserved residues were placed into the catalytic portion of the hammerhead (Haseloff and Gerlach, 1988). The requirements for trans -cleavage by hammerhead ribozymes include a UH (where H is an A, C, or U residue) recognition site in the target RNA and the ability to base pair with the target (Koizumi et al, 1988;Ruffner et al, 1990). The base-pairing region, or arms, provides specificity to direct the catalytic domain of the ribozyme to the target site of the substrate RNA.…”