Browsing by Author "Dr. Michael Hyman, Committee Member"

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Characterization of In Vitro Selected RNA Aptamers for the RB69 RegA Translational Repressor Protein and Optimization of in vitro Selection Utilizing Complex Targets
(2003-01-30) Dean, Timothy Richard; Dr. Dennis Brown, Committee Member; Dr. Eric Miller, Committee Chair; Dr. Michael Hyman, Committee Member; Dr. James Brown, Committee Member
The purpose of the research has been to characterize the nucleic acid sequence requirements necessary for recognition by the translational repressor protein RegA. RegA, from bacteriophage RB69 and T4 related bacteriophages, is a translational repressor protein that is highly conserved and capable of repressing translation of numerous transcripts. The mRNA binding sites for RegA are devoid of structure and are AU rich. in vitro selection was carried out for 5 rounds generating specific sequences capable of binding to RB69 RegA. Characterization of those sequences included sequencing, dissociation constant (Kdapp) determination, and use of the selected sequences to search the RB69 genome for potential binding sites. The second aspect of the research focused on optimization of conditions for completing successful in vitro selection experiments against complex biological targets. Bacteriophage R17 is an F+ coliphage infecting E. coli found in the intestinal tracts of swine. Development of a rapid diagnostic technique capable of identifying R17 would enable for detection of faeces in ground water. Generation of RNA aptamers against R17 was undertaken with the goal of optimizing selection conditions against a viable viral particle. Two selections were carried out one utilizing nitrocellulose to partition bound RNA from unbound RNA and the second using R17 immobilized on a agarose bead matrix. The results initiated the generation of specific considerations for initiating a selection experiment against a protein target.