In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme.
We have previously reported the development of a group I intron-derived ribozyme that can bind an exogenous RNA substrate and excise from that substrate an internal segment in vitro, which allows for sequence-specific modification of RNA molecules. In this report, the activity of this trans excision-splicing ribozyme in a cellular environment, specifically Escherichia coli, was investigated. The ribozyme was re-engineered to target for excision a single-base insertion in the transcript of a green fluorescent protein, and fluorescence was exploited as a reporter for trans excision-splicing. We show that the ribozyme is able to catalyze the trans excision-splicing reaction in vivo and can repair the mutant transcripts. On average, 12% correction is observed as measured by fluorescence and at least 0.6% correction as confirmed through sequence analysis. This represents the first report of a biomolecule (in this case a ribozyme) that can selectively excise a targeted nucleotide from within an mRNA transcript in vivo. This new class of biochemical tools makes possible a wide variety of new experimental strategies, perhaps including a new approach to molecular-based therapeutics.
Baum
Dana A
DA
Department of Chemistry, University of Kentucky, Lexington, 40506, USA.
Testa
Stephen M
SM
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007438:Introns; D009154:Mutation; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*; D020040:Trans-Splicing; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012265:Ribonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins