Addition of an extra substrate binding site and partial destabilization of stem structures in HDV ribozyme give rise to high sequence-specificity for its target RNA.
Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.
Hori
Tamaki
T
Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama, Japan.
Guo
Fei
F
Uesugi
Seiichi
S
D001483:Base Sequence; D001665:Binding Sites; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012313:RNA