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The Catholic University of America THE CATHOLIC UNIVERSITY OF AMERICA Development and Use of Novel Transverse Magnetic Tweezers for Single-Molecule Studies of DNA-Protein Interactions A DISSERTATION Submitted to the Faculty of the Department of Biomedical Engineering School of Engineering Of The Catholic University of America In Partial Fulfillment of the Requirements For the Degree Doctor of Philosophy By Christopher D. Tyson Washington, D.C. 2016 Development and Use of Novel Transverse Magnetic Tweezers for Single-Molecule Studies of DNA-Protein Interactions Christopher D. Tyson, Ph.D. Director: Abhijit Sarkar, Ph.D. I describe several contributions to single molecule experiments. A transverse magnetic tweezers is presented that enables in-plane micromechanical manipulation of a single DNA molecule. This includes a new method for tethering DNA utilizing two labeled beads and a functionalized glass micro-rod. The attachment chemistry reported here enables rapid capture of multiple DNA tethers in parallel, overcomes the difficulties associated with bead aspiration, and preserves the ability to perform differential extension measurements from the bead centroids. Combined with micro- injection pipettes, a new sample cell design, and a buffer exchange system, the components increase the ease-of-use and experimental throughput of the magnetic tweezers device. On the software side, several unique computational methods for interrogating single molecule data are described. First, a technique that uses the diffraction pattern of beads to perform sub-pixel, ~10 nm-level localization of the bead centroids is explained. Second, a novel method for automatically detecting steps in DNA extension data is presented. This algorithm is well-suited for analyzing experiments involving binding and force-induced unbinding of DNA-protein complexes, which produce flat extension regions – steps – corresponding to the times between individual protein association or dissociation events. Finally, a new algorithm for tracking densely-populated, fast spawning, indistinguishable objects moving unidirectionally at high-velocities is developed and its performance thoroughly characterized. Together, these results should improve single molecule micromanipulation techniques by providing a hardware and software combination that can be implemented and used relatively easily, while enabling near-Brownian-noise limit force and extension measurements on DNA and DNA-protein complexes. This dissertation by Christopher D. Tyson fulfills the dissertation requirement for the doctoral degree in Biomedical Engineering approved by Otto Wilson, Ph.D., as Advisor, and by Abhijit Sarkar, Ph.D., and Lorenzo Resca, Ph.D. as Readers. _________________________________________ Otto Wilson, Ph.D., Advisor _________________________________________ Abhijit Sarkar, Ph.D., Reader _________________________________________ Lorenzo Resca, Ph.D., Reader ii Acknowledgements For their contributions in support of my work, I would like to thank: Roberto Fabian Christopher McAndrew, Ph.D. Anneliese Striz Prof. Pamela Tuma, Ph.D. Prof. Ian L. Pegg, Ph. D. Additionally, funding from the Vitreous State Laboratory and The Catholic University of America is gratefully acknowledged. iii Table of Contents Chapter 1: Introduction 1.1 Single-molecule Biology 1.2 DNA 1.3 Proteins 1.4 Response of DNA to Micromechanical Manipulation 1.5 Studying DNA-Protein Interactions with Single Molecule Methods 1.6 Research Problem and Approach 1.7 Plan of Research Chapter 2: Transverse Magnetic Tweezers 2.1 Introduction to Methods for Single-molecule Micromechanical Manipulation 2.2 Horizontal Magnetic Tweezers Methodology 2.2.1 Attachment Protocol 2.2.2 Horizontal Magnetic Tweezers Device Design 2.2.3 Optical Calibration with Graticule 2.2.4 Force Calculation via Fluctuation-dissipation Theorem 2.2.5 Force Calibration via Stokes Law 2.3 Experimental Results iv 2.3.1 DNA tether extension 2.3.2 Force Calibration results 2.3.3 Determination of Experimental Precision 2.3.4 DNA-protein complexation 2.4 Discussion Chapter 3: Step-finding Algorithm 3.1 Introduction to Step-finding Algorithms 3.2 Step-finding Algorithm Methodology 3.2.1 Step-finding Algorithm description 3.2.2 Step-trace simulations 3.2.3 Performance Analysis 3.2.4 Experimental Data 3.3 Results 3.3.1 Simulation Results 3.3.2 Algorithm Performance 3.4 Discussion Chapter 4: Object Tracking Algorithm v 4.1 Introduction to Tracking Algorithms 4.2 Object Tracking Algorithm Methodology 4.2.1 Tracker Algorithm Description 4.2.2 Trajectory Simulations 4.2.3 Performance Analysis 4.2.4 Experimental Data 4.3 Results 4.3.1 Total Number of Objects 4.3.2 Initial x-position 4.3.3 Initial y-position 4.3.4 Initial x-velocity 4.3.5 Initial y-velocity 4.3.6 y-acceleration 4.3.7 Noise and Spawn rate 4.3.8 Experimental Parameter Simulations 4.4 Discussion Chapter 5: Conclusions 5.1 Summary of Results vi 5.2 Future Directions Appendices A.1 Protocol for surface functionalization of glass micro-rod A.2 Protocol for DNA End-functionalization A.3 Protocol for labeling of superparamagnetic beads with anti-digoxigenin A.4 Magnetic Tweezers Component List A.5 Protocol for preparation of histones A.6 Bead Centroid Localization code A.7 Force Calculation code A.8 Step-generator Simulation code A.9 Step-finding Algorithm code A.10 Object Tracking code A.11 Trajectory Simulation code vii Chapter 1 - Introduction 1.1 Single Molecule Techniques – An Overview Single molecule techniques involve high precision measurements on mechanical and optical signals from individual biological macromolecules in vitro and inside living cells, in vivo. Traditionally, proteins and DNA have been studied using ensemble techniques that involve large numbers of molecules. The resulting data are population averages, making it difficult to infer information about the distributions around mean molecular responses or to detect and characterize unusual but biologically-relevant subpopulations of molecules. Single molecule techniques are powerful because they provide a means to overcome these limitations. For instance, using single molecule methods, rare or short-lived intermediate macromolecular conformations that would otherwise be averaged out in an ensemble assay can be discovered and thoroughly investigated. When protein-DNA interactions display heterogeneity in their kinetics, single molecule approaches are well-suited to distinguish alternate reaction pathways. In ensemble assays, on the other hand, very large number of molecules would have to have their activities synchronized to obtain comparable data, something nearly impossible to do in those experiments (Nahas et al. 2004, 1107-1113). Moreover, direct measurement of displacements, torques, twists, forces, and free energies of interacting 1 2 biomacromolecules is only possible at the single molecule level. Another advantage is that single molecule approaches naturally take into account the role of fluctuations in in vivo processes. Fluctuations arise because biopolymers are sometimes present inside cells in low copy numbers, thus giving concentration fluctuations a large role (Ghaemmaghami et al. 2003, 737-741). Figure 1.1 shows a conceptual comparison between ensemble assays (1.1A) and single molecule (1.1B) approaches. Figure 1.1 Comparison between Ensemble Assays and Single-molecule Techniques. (A) Ensemble assays observe multiple events simultaneously, leading to a signal which is an average over individual events. In this case, the photon counts from a large number of fluorescent proteins produce a noisy, averaged signal. (B) A single molecule approach allows individual fluorescence on- and off-states to be observed. 3 (Image courtesy Arrondo, Jose Luis R., and Alicia Alonso. 2006. Advanced Techniques in Biophysics. Berlin Heidelberg: Springer-Verlag.) Single molecule methods can be divided into two categories: fluorescence microscopy and force spectroscopy. Fluorescence techniques involve the imaging of single fluorescent molecules, fluorophores, at room temperature. The key challenge here is the very low photon signal-to-noise ratio that makes detection, imaging, and localization of individual, densely-clustered fluorophores extremely technically demanding. Furthermore, these experiments must be performed with samples kept at room temperature, in aqueous buffers, and at high-enough acquisition rates to assay fast DNA and protein conformational fluctuations. However, recent advances in (a) microscope objective lens designs, (b) charged-coupled device (CCD) and complementary metal oxide semiconductor (CMOS) detectors, (c) fluorescent dyes, (d) protein- and DNA-dye attachment chemistries, (e) computational algorithms, and (f) experimental designs have now enabled three dimensional, sub-diffraction-limit detection, localization, and imaging of individual, densely-clustered fluorophores in biologically-relevant contexts (Brooks Shera et al. 1990, 553-557; Betzig and Chichester 1993, 1422-1425). 4 Force spectroscopy techniques are used to investigate the micromechanical responses of biomacromolecules, mainly DNA and DNA-protein complexes. The general approach involves tethering a single linear DNA molecule to a glass coverslip at one end and a micron-sized polystyrene bead at the other end. An important variation involves replacing the coverslip with a second bead resulting in dual-bead tethers. The beads act as macroscopic handles that can be manipulated in a variety of ways. The major micromanipulation approaches use (a) optical traps, (b) magnetic tweezers (or magnetic traps), (c)
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