Single-molecule methods for an improved understanding of biophysical interactions: from fundamental biology to applied nanotechnology

Single-molecule;carbon nanotube;protein-DNA interactions;nanotechnology;nanotoxicity;force spectroscopy;Fluorescence Resonance Energy Transfer (FRET);fluorescence microscopy;target search;optical traps;oxidative damage;singlet oxygen

Landry, Markita

关键词: Single-molecule;    carbon nanotube;    protein-DNA interactions;    nanotechnology;    nanotoxicity;    force spectroscopy;    Fluorescence Resonance Energy Transfer (FRET);    fluorescence microscopy;    target search;    optical traps;    oxidative damage;    singlet oxygen;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/42430/Markita_Landry.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The advent of single-molecule methods has greatly extended the scale at which we are able to probe natural systems. The information that can be gained by studying biological systems on a single-molecule scale, in the absence of ensemble averaging, provides an unprecedented amount of detail about molecular interactions in real-time. Single-molecule biophysical techniques have provided unique insights into the nature of protein-DNA interactions, and have allowed for the development of novel platforms to study nano-bio interactions. In this thesis, we will describe two main sets of experiments to explore molecular interactions at the single-molecule scale. We will focus on the study of protein-DNA interactions and also the interactions between biological molecules and synthetic nanoparticles, using a variety of single-molecule techniques.Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization or oligomerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. Interestingly, we observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations of dimer-active protein search, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins (SSPs). In addition to studying the target-search process of protelomerase TelK, we have also studied the molecular mechanism of TelK activity at the target site. We attempt to capture the dynamics responsible for DNA hairpin formation by TelK, and we discuss the unique features of TelK-DNA interactions that contribute to the complexity of this process.Nanomaterials have unique optical, chemical and mechanical properties that make them useful in biological applications, acting as drug and gene delivery agents, electrical and optical sensors, and cell-signaling components. Although many tools exist to characterize both biomolecules and nanomaterials, these methods are currently unable to give a detailed picture of biomolecular structure at the nano-bio interface. As a result, local electronic properties, bioavailability, toxicological effects, and basic molecular structure and conformation of biomolecules on nanoparticles remain unclear.Single-Walled Nanotubes (SWNTs) are allotropes of carbon with a cylindrical nanostructure. Though SWNTs tend to form insoluble aggregates, sonicating SWNTs with DNA forms a DNA-SWNT complex that is soluble in water. Single-stranded DNA (ssDNA) is believed to form a helical structure on the SWNT surface. This DNA-SWNT complex is not only soluble in water and does not appear to be toxic to mammalian cells, but it is also uptaken by mammalian cells via endocytosis. Therefore, there is significant interest in understanding the mechanism of SWNT encapsulation by ssDNA. However, current experimental tools have been unable to probe the structure of biomolecules on the surface of nanomaterials. Consequently, little is known about the mechanism by which ssDNA wraps SWNT, and how biomolecules interact with the resulting DNA-SWNT structure. In order to extend the range of biochemical interactions that can be detected on a SWNT surface, we have developed a variety of experimental platforms to study biological interactions on SWNT surfaces by extending several well-established single-molecule biophysics techniques to the study of nano-bio interactions. By applying single-molecule techniques to the study of the nano-bio interface, we uncover changes in the expected behavior of biomolecules. These effects include cooperative DNA hybridization, changes in the accessibility of DNA to nuclease proteins, and protein deactivation on a SWNT surface. We also uncover details of the mechanism by which ssDNA wraps SWNT to form a biologically-compatible nanoparticle-biomolecule conjugate.