The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA.
The catalytic center of the RNA from the negative strand of the satellite RNA of chicory yellow mottle virus type 1 (sCYMV1) is in the hairpin ribozyme family, has catalytic activity, and cleaves substrates before a preferred GUA sequence. This is different from that of the satellite RNA from the negative strand of tobacco ringspot virus (sTRSV) which prefers a GUC sequence at the site of cleavage. The sCYMV1 hairpin ribozyme has now been developed for cleaving heterologous RNA substrates. When helix 1 was extended from the native 5 bp to 6 bp with a newly added A:U base pair, catalytic activity increased three-fold. The preferred sequence for the substrate loop was the native A*GUA sequence where * is the site of cleavage. When each nucleotide in this sequence was changed to each of the other three nucleotides, catalytic activity decreased 66-100%. RNA targets, containing this A*GUA sequence, were located in both human papillomavirus and HIV-1. Ribozymes were developed which efficiently cleaved these targets in vitro. These results identify a new class of hairpin ribozymes capable of cleaving substrates before a preferred GUA sequence rather than the GUC preferred by the sTRSV hairpin ribozyme. This expands the repertoire of target sites available for gene therapy using the hairpin ribozyme.
Lian
Y
Y
Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA.
De Young
M B
MB
Siwkowski
A
A
Hampel
A
A
Rappaport
J
J
D018390:Gene Targeting; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D009029:Mosaic Viruses / Q000235:genetics*; D016296:Mutagenesis / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics; D010942:Plant Viruses / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*
D016337:RNA, Catalytic; D012367:RNA, Viral