Exploring Novel Techniques for the Single Molecule Toolkit: Vesicle Encapsulation and Immobilization
Okumus, Burak
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https://hdl.handle.net/2142/85457
Description
Title
Exploring Novel Techniques for the Single Molecule Toolkit: Vesicle Encapsulation and Immobilization
Author(s)
Okumus, Burak
Issue Date
2006
Doctoral Committee Chair(s)
Ha, Taekjip
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Physics, Molecular
Language
eng
Abstract
Tracking asynchronous time evolution of single biological molecules provides unique insights into detailed reaction kinetics and pathways. Such measurements are frequently made on macromolecules that are tethered on a glass surface. However, there have been reports of variability of surface environment, and suspicion that observed heterogeneity of dynamic properties in single molecules might be an artifact of the local surface. A striking example is the hairpin ribozyme which was shown---in our lab---to exhibit two orders of magnitude variation in folding/unfolding kinetics between molecules. Moreover, a DNA with a sequence of human telomeric repeat exhibited extreme conformational diversity among six interconverting conformations. In order to find out the true nature of the observed heterogeneities, we encapsulated the ribozyme and the human telomeric DNA inside liposomes (i.e. artificially formed phospholipid vesicles) which were then tethered on the surface. Our data revealed similar behavior for encapsulated and the conventionally attached nucleic acid molecules. Although vesicle encapsulation offers a biologically relevant environment for many soluble proteins and nucleic acids, impermeability towards ions and other small molecules such as ATP hinders more general applications. We therefore developed methods to induce pores into vesicles which open up the possibility of using them as ultra-small, bio-mimetic, porous containers. Porous vesicles were then utilized to perform unique measurements for observing RecA filament formation, hairpin ribozyme cleavage and Rep helicase translocation within confined volumes. Novel features were revealed by such experiments unveiling new biological findings. We also discuss ideas to introduce pores that can be opened up via ultraviolet radiation for future applications. Aside from the encapsulation studies, we developed a new assay to detect the SNARE mediated membrane fusion by using surface attached proteliposomes. Such an approach is not only able to dissect the intermediates on the membrane fusion pathway, but also provides a general and bio-friendly platform for the surface tethering of membrane systems. We anticipate that the vesicle encapsulation and immobilization methods will greatly expand the horizon of single-molecule measurements and will transform the way single-molecule fluorescence measurements are performed in many laboratories around the world.
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