IN VITRO AND IN VIVO CHARACTERIZATION OF A TRANS EXCISION-SPLICING RIBOZYME

• Main Author: Baum, Dana Ann
• Format: Others
• Published: UKnowledge 2005
• Subjects: Group I intron|ribozyme|trans excision-splicing|splice site sequence requirements|in vivo targeted nucleotide excision
• Online Access: http://uknowledge.uky.edu/gradschool_diss/284; http://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1287&context=gradschool_diss

Group I introns are catalytic RNAs with the ability to splice out of RNA transcripts, often without the aid of proteins. These self-splicing introns have been reengineered to create ribozymes with the ability to catalyze reactions. One such ribozyme, derived from a Pneumocystis carinii group I intron, has been engineered to sequence specifically remove a targeted segment from within an RNA substrate, which is called the trans excision-splicing reaction.The two catalytic steps of the trans excision-splicing reaction occur at positions on the substrate known as the 5' and 3' splice sites. Strict sequence requirements at these sites could potentially limit the target choices for the trans excision-splicing ribozyme, so the sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. All base pair combinations at the 5' splice site allow the first reaction step (5' hydrolysis) to occur and several combinations allow the second step to occur, resulting in trans excision-splicing product formation. Moreover, we found that non-Watson-Crick base pairs are important for 5' splice site recognition and prevent product degradation via hydrolysis at other sequence positions. The sequence requirement at the 3' splice site is absolute, as guanosine alone produced complete product.To date, the experiments with the trans excision-splicing ribozyme have been conducted in vitro. The further development of this ribozyme as a biochemical tool and as a potential therapeutic agent requires in vivo reactivity. Thus, a prokaryotic system was designed and tested to assess the catalytic potential of the trans excision-splicing ribozyme. We show that the ribozyme successfully excised a single, targeted nucleotide from a mutated green fluorescent protein transcript in Escherichia coli. On average, 12% correction was observed as measured by fluorescence and approximately 1.2% correction was confirmed through sequence analysis of isolated transcripts.We have used these studies to further characterize trans excision-splicing ribozymes in vitro and to pave the way for future development of this ribozymereaction in vivo. These results increase our understanding of this ribozyme and advance this reaction as a biochemical tool with potential therapeutic applications.