A Computational Study of DNA Four-way Junctions and Their Significance to DNA Nanotechnology by Matthew R. Adendorff BSc. (Hons), MSc. Chemistry Rhodes University, 2010 SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL ENGINEERING IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGICAL ENGINEERING AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2016 © Massachusetts Institute of Technology, 2016. All rights reserved. Signature of Author: ____________________________________________________________ Department of Biological Engineering December 18, 2015 Certified by: __________________________________________________________________ Mark Bathe Associate Professor of Biological Engineering Thesis Supervisor Accepted by: _________________________________________________________________ Forest White Professor of Biological Engineering Chairman, Graduate Program Committee Thesis Committee: Professor Bruce Tidor Professor of Biological Engineering and Computer Science Massachusetts Institute of Technology Thesis Committee Chair Professor William Shih Associate Professor of Biological Chemistry and Molecular Pharmacology Harvard University Thesis Committee Member 2 A Computational Study of DNA Four-way Junctions and Their Significance to DNA Nanotechnology by Matthew R. Adendorff Submitted to the Department of Biological Engineering on December 18, 2015, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering ABSTRACT The field of DNA nanotechnology has rapidly evolved over the past three decades, reaching a point where researchers can conceive of and implement both bioinspired and biomimetic devices using the programmed self-assembly of DNA molecules. The sophisticated natural systems that these devices seek to interrogate and to imitate have Angstrom-level organizational precision, however, and the nanotechnology community faces the challenge of fine-tuning their design principles to match. A necessity for achieving this level of spatial control is an understanding of the atomic-level physico-chemical interactions and temporal dynamics inherent to fundamental structural motifs used for nanodevice design. The stacked configurational isomers of four-way junctions, the motif on which DNA nanotechnology was founded, are the focus of this work; initially in isolation and then as part of larger DNA nano-assemblies. The first study presented here investigates the impact of sequence on the structure, stability, and flexibility of these junction isomers, along with their canonical B-form duplex, nicked-duplex and single cross-over topological variants. Using explicit solvent and counterion molecular dynamics simulations, the base-pair level interactions that influence experimentally-observed conformational state preferences are interrogated and free- energy calculations provide a detailed theoretical picture of isomerization thermodynamics. Next, the synergy of single molecule imaging, computational modelling, and a novel enzymatic assay is exploited to characterize the three-dimensional structure and catalytic function of a DNA tweezer-actuated nanoreactor. The analyses presented here show that rational redesign of the four-way junctions in the device enables the tweezers to be more completely and uniformly closed, while the sequence-level design strategies explored in this study provide guidelines for improving the performance of DNA-based structures. Finally, MD simulations are used to inform finite-element method coarse-grained models for the ground-state structure determination and equilibrium Brownian Dynamics of large-scale DNA origamis. Together, this thesis presents a set of guidelines for the rational design of nanodevices comprising arrays of constrained four-way junctions. Thesis Supervisor: Mark Bathe Title: Associate Professor of Biological Engineering 3 4 ACKNOWLEDGEMENTS I would first like to thank my advisor Prof. Mark Bathe for the years of guidance and mentorship that he has given to me. In his lab I have grown into a competent scientist and have learned from him how to identify and to tackle research problems with a systematic rigor that is built on a foundation of sound literature study. I thank my thesis committee chair Prof. Bruce Tidor for his invaluable input over the years and for helping me to ask critical, focused research questions. I thank my committee member Prof. William Shih for his knowledgeable guidance and for helping me to identify significant research questions that can benefit the DNA nanotechnology community. I am also grateful to the Army Research Office, the Office for Naval Research, and the International Science and Technology Fulbright Program for funding over the course of my PhD. Significant thanks are due to Keyao Pan who has been both a teacher and a friend to me during the many projects we have worked on together. I also thank members of the Bathe Lab, both former and current, for being such excellent and knowledgeable colleagues: Zach Barry, Remi Veneziano, Sakul Ratanalert, Aprotim Mazumder, Will Bricker, Tyson Shepherd, Stavros Gaitanaros, Etienne Boulais, Do-Nyun Kim, Reza Sharifi Sedeh, Philip Bransford, Syuan-Ming Guo, Simon Gordonov, Philipp Diesinger, Hyungmin Jun, Yera Hakobyan, Nilah Monnier, Larson Hogstrom, and Darlene Ray for always taking such good care of us. Special thanks must go to Prof. Doug Lauffenburger and the Biological Engineering department, especially: Anthony “Toro” Soltis, Chris “Richard” Ng, Jorge “George” Valdez, Shannon Hughes, the BE PhD class of 2015, Susan Jaskela, Aran Parillo, Dan Darling, and Dalia Fares. I thank the Simmons Hall team, in particular: Tyler and Dana Susko, Tim Milakovich, Hans Rinderknicht and Mariah Steele, Josh Gonzalez, Nika Hollingsworth, Steve Hall, and John and Ellen Essigmann. It has been an honour to be a part of the MIT Rugby family and to play with such fantastic gentlemen during my tenure at MIT, thank you and go Tech! Significant personal thanks are due to Adam Labadorf, Zach Boswell, Jarrad Langley, my nemesis Nick Allard, Tom Nyilis, Patrick Goulet, Liz Plageman and Dave Chaet, Lauren Melby and Zach Gerson-Nieder, Meaghan Texeira, Ognjen and Nicole Ilic, Dave and Natalia Worth, Phil and Aimee Brierley, Richard Gevers, and Heiko Heilgendorff. Thank you to my loving family: Emily Adendorff, Ralph and Trudi Adendorff, Shara and David Matlin, and Rox and Stu Lasky. Finally, to my wonderful wife Ally, thank you for all your immense love and support; this thesis is dedicated to you 5 "Because learning does not consist only of knowing what we must or we can do, but also of knowing what we could do and perhaps should not do." - Umberto Eco 6 TABLE OF CONTENTS A. GENERAL INTRODUCTION .................................................................................................. 11 1.1. Fundamentals of DNA nanotechnology ........................................................................ 11 1.1.1. Origins ................................................................................................................... 11 1.1.2. Milestones in structural DNA nanotechnology ........................................................ 12 1.1.3. Computational design of DNA nanostructures ........................................................ 13 1.1.4. Improving spatial control ........................................................................................ 14 1.2. Modelling approaches to DNA ....................................................................................... 15 1.2.1. Statistical models ................................................................................................... 16 1.2.2. Thermodynamic Models ......................................................................................... 17 1.2.3. Atomistic models .................................................................................................... 17 1.2.4. Continuum Models ................................................................................................. 19 1.2.5. Unbiased MD formulation ....................................................................................... 20 1.2.6. Essential dynamics analysis .................................................................................. 21 1.2.7. Collective variable analysis .................................................................................... 23 1.2.8. Helical parameter analysis ..................................................................................... 25 1.2.9. Free-energy calculations ........................................................................................ 26 1.2.9.1. Replica-based methods .............................................................................. 27 1.2.9.2. Biasing potential methods .......................................................................... 28 1.2.9.3. The MM-PBSA method ............................................................................... 30 1.3. Overview of thesis contents .......................................................................................... 31 2. SEQUENCE EFFECTS IN IMMOBILE DNA FOUR-WAY JUNCTIONS: A COMPUTATIONAL STUDY .................................................................................................... 33 2.1. Introduction .................................................................................................................... 34 2.2. Materials and Methods ..................................................................................................