Dissertation
A proton-sensitive ribozyme switch with "molecular memory"
Doctor of Philosophy (Ph.D.), Drexel University
Mar 2003
DOI:
https://doi.org/10.17918/00000502
Abstract
The self-cleaving hammerhead ribozyme has been re-engineered to function as an allosteric switch that is sensitive to pH. Through in vitro selection of an RNA population consisting of the hammerhead motif with the stem-loop II domain randomized, a population of active self-cleaving ribozymes was enriched after six rounds of selection. The ribozymes turn "on" only when they are initially pulsed with acid at or below pH 4.6. Subsequent neutralization in the presence of magnesium ions allows self-cleavage to occur with kinetics that approach the maximum wildtype activity of the hammerhead ribozyme ( kcat = 1 min-1). In its uninduced state, the ribozyme exhibits a background rate constant (k uncat < 0.0006 min-1). The study of a representative clone reveals that the acidic environment rapidly activates the ribozyme ( k1 > 1 min-1) manifested by an allosteric structural change into an active conformation. However, it is also shown that in the absence of divalent cations, the activated ribozyme rapidly reverts back to an inactive state (k-1, > 1 min -1). Because the rate of inactivation exceeds the rate of catalysis, it is likely that magnesium plays a structural role in stabilizing the "on" state in addition to its role in hammerhead catalysis. Further evidence shows that by replacing magnesium with a similar divalent, Sr+2, which is catalytically inert, the ribozyme is stable in the "on" state even after neutralization for up to 60 minutes. This 'molecular memory' effect, in which the ribozyme 'remembers' that it encountered an acidic medium, has been enhanced by performing several more rounds of selection for increased memory. Clones from generation eleven populations exhibit molecular memory for >24 hours. These findings suggest that RNA switches and other engineered RNAs could be made to serve as molecular components of advanced molecular systems.
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Details
- Title
- A proton-sensitive ribozyme switch with "molecular memory"
- Creators
- Gaetano T. Caltagirone
- Contributors
- Richard F. Rest (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- ix, 163 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Medicine; Neurology; Drexel University
- Other Identifier
- 991014970316704721