Single Molecule Fluorescence Imaging of Biosensors, Ribozymes and Molecular Spiders.

Abstract

Single molecule fluorescence imaging has been developed in recent times to expand our understanding of the heterogeneity and biological mechanisms of molecular ensembles. In this dissertation, such imaging techniques, along with ensemble fluorescence spectroscopy tools have been used to investigate three systems of nucleic acid enzymes.

An engineered biosensor built from a theophylline aptamer and the hammerhead ribozyme (termed an aptazyme) was scrutinized using single molecule fluorescence resonance energy transfer (smFRET) and ensemble fluorescence studies. It was found that a catalytically active state is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to the limited dynamic range (<100 fold) observed for the aptazyme. In addition, slow conformational rearrangements dampen the speed in which the catalytically active conformation is accessed. In contrast, the only known naturally occurring aptazyme uses a chemical cofactor to instantaneously trigger catalysis (with a 100,000 fold activation range), rather than the slower rearrangement of an inactive into an active structure.

To examine the effects of the U-turn of the hepatitis delta virus (HDV) ribozyme, recently found to be at the heart of the ribozyme’s catalytic core, both DNA and RNA ligase mediated methods were evaluated to assemble the ribozyme from chemical synthesized fragments. Upon successful assembly of the ribozyme, preliminary smFRET studies were performed revealing global dynamics and heterogeneity promising to unveil new insight into the functional role of the U-turn.

Recently designed nano-robots called Molecular Spiders use nucleic acid enzymes as “fuel” to traverse on a specific two-dimensional landscape. In this thesis, individual Spider movement was surveyed by single fluorescent particle tracking. Two-dimensional Spider movement was followed in real-time, providing evidence for the previously hypothesized model that Spiders move in a self-repellent autonomous (cybernetic) walk. These nano-walkers represent potential drug delivery vehicles with the ability to understand and follow external cues.

Overall, the work presented in this dissertation has illuminated the suitability of single particle fluorescence techniques to monitor the functional behavior and heterogeneity of single nucleic-acid based molecules ranging from biosensors and small catalytic ribozymes to novel molecular nano-robots.