Photochemical Sciences Ph.D. Dissertations

Design and Characterization of Novel Bio-Sensor Platform for Sequence Specific, Label-Free, Fluorescent Detection of Native RNA Moledcules

Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

Neocles B. Leontis

Second Advisor

Jeffrey A. Snyder (Committee Member)

Third Advisor

R. Marshall Wilson (Committee Member)

Fourth Advisor

Michael A. Rodgers (Committee Member)

Abstract

This project describes a new bio-sensor platform for sequence-specific, label-free,fluorescent detection of pre-folded RNA molecules. This detection is based on paranemic association of a sensor RNA molecule to a target RNA molecule using Watson-Crick basepairing. We demonstrate the paranemic association can be accomplished with a minimal 3-half-turn (3HT) paranemic pairing motif. The motif results from two strand exchanges between the sensor and target RNAs that allow for the formation of 10 to 16 inter-molecular Watson-Crick basepairs in major (M) groove or 8 to 14 inter-molecular Watson-Crick basepairs in minor (m) groove when the sensor and target molecules have complementary sequences. Paranemic association does not require unfolding of preformed secondary structures in either the sensor or target molecules. This poject teaches how to position and orient an aptamer for the triphenylmethane dye Malachite Green (MG) within the sensor RNA so that the sensor RNA only binds MG at the aptamers site when it is bound in turn to the target RNA. When the sensor/target complex forms, it binds MG at the aptamer site and the MG becomes fluorescent and thus signals the presence of the target RNA. In the absence of the target RNA, the sensor RNA is not able to bind MG, so the MG remains free in solution and no fluorescence is observed. Thus the system performs as a fluorescent sensor for the target RNA without the need to covalently attach a fluorescent moiety to either the sensor or the target.

This fluorescent sensor system also has the potential to be used as an RNA-chromophore-assisted laser inactivation (RNA-CALI) agent providing light-induced degradation of the sensor/target complex.

Also in this dissertation proposal we investigate how to modulate the helical twist of RNA molecules using C-loop, a new modular recurrent RNA motif, recently identified in crystal structures, to generate a new, specific self-assembly interface, using known cognate loop-receptor motifs. This design is intended for use in a second biosensor platform design that employs specific loop-receptor interactions and is still under development. Furthermore, these results shed new light on possible roles for these motifs in biological structures.

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