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

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.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62342/1/chamaree_1.pd

Utilization of Optical Tweezer Nanotechnology in Membrane Interaction Studies

Optical tweezers have been a fixture of microscopic cell manipulation since the 1990s. Arthur Ashkin&rsquo;s seminal work has led to the advancement of optical tweezers as an effective tool for assay development in the fields of physics and nanotechnology. As an advanced application of cell manipulation, optical tweezers have facilitated the study of a multitude of cellular and molecular interactions within the greater field of nanotechnology. In the three decades since the optical tweezers&rsquo; rise to prominence, different and versatile assays have emerged that further explore the biochemical pathways integral for cell proliferation and communication. The most critical organelle implicated in the communication and protection of single cells includes the plasma membrane. In the past three decades, novel assays have emerged which examine the plasma membrane&rsquo;s role in cell-to-cell interaction and the specific protein components that serve integral membrane functions for the cell as a whole. To further understand the extent to which optical tweezers have evolved as a critical tool for cellular membrane assessment within the field of nanotechnology, the various novel assays, including pulling, indentation, and stretching assays, will be reviewed in the current research sector

Utilization of Optical Tweezer Nanotechnology in Membrane Interaction Studies

Optical tweezers have been a fixture of microscopic cell manipulation since the 1990s. Arthur Ashkin’s seminal work has led to the advancement of optical tweezers as an effective tool for assay development in the fields of physics and nanotechnology. As an advanced application of cell manipulation, optical tweezers have facilitated the study of a multitude of cellular and molecular interactions within the greater field of nanotechnology. In the three decades since the optical tweezers’ rise to prominence, different and versatile assays have emerged that further explore the biochemical pathways integral for cell proliferation and communication. The most critical organelle implicated in the communication and protection of single cells includes the plasma membrane. In the past three decades, novel assays have emerged which examine the plasma membrane’s role in cell-to-cell interaction and the specific protein components that serve integral membrane functions for the cell as a whole. To further understand the extent to which optical tweezers have evolved as a critical tool for cellular membrane assessment within the field of nanotechnology, the various novel assays, including pulling, indentation, and stretching assays, will be reviewed in the current research sector

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme 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 a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer