Deriving a 67-nucleotide trans-cleaving ribozyme from the hepatitis delta virus antigenomic RNA.
RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and produced Ag5-84, which cleaved to 100% at 37 degrees C in less than 0.25 min. Internal deletions of the Stem IV region resulted in Ag5-73, still capable of efficient cleavage, although with a lessened stability in formamide. A trans-cleaving enzyme-substrate pair was finally derived from this RNA, and it consisted of a 67-nucleotide enzyme that cleaved a 13-nucleotide RNA substrate.
Prasad
Y
Y
Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania.
Smith
J B
JB
Gottlieb
P A
PA
Bentz
J
J
Dinter-Gottlieb
G
G
D001483:Base Sequence; D016679:Genome, Viral; D003698:Hepatitis Delta Virus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016372:RNA, Antisense / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D016372:RNA, Antisense; D016337:RNA, Catalytic; D012367:RNA, Viral