Developing and Understanding Nucleic Acid Tension Sensors to Dissect Cellular Mechanosensing Público
Shinde, Pushkar (Spring 2022)
Abstract
Five chapters comprise this thesis. The first provides a motivation for studying mechanobiology and introduces the tools and techniques: Molecular Tension Fluorescence Microscopy and nucleic acid tension probes. The second chapter explores the relationship between nucleic acid tension probe structure and function through molecular dynamics simulations. These data suggest that subtle sequence variations lead to significant structural alterations that can tune probe photophysical properties without significantly affecting mechanics. The third chapter designs and explores a mathematical model of a set of nucleic acid tension probes to measure bond lifetime. The results suggest these probes may be feasible and outlines several challenges that would need to be overcome to facilitate their implementation. The fourth chapter develops probes with the same force to open but dramatically different work thresholds, to understand the fundamental mechanisms of T-cell triggering. The results suggest that the T-Cell Receptor (TCR) responds to more than force, though further experimentation is required to understand exactly what the TCR senses. The final chapter briefly summarizes the work and places it in a larger context. Holistically, this work aims to help advance mechanobiology by exploring the relationships between tension probe structure, the physical parameters to which these probes respond, the mechanical stimulus these probes present receptors, and receptors’ responses to those stimuli.
Table of Contents
Chapter 1: Introduction 1
Chapter 2: Understanding Tension Probe Structure and Function 6
INTRODUCTION 7
RESULTS 9
+ Spacer Hairpins 11
- Spacer Hairpins 13
DISCUSSION 14
Effect of Structure on Mechanics 14
Structure and Photophysics 16
CONCLUSIONS AND NEXT STEPS 18
METHODS 19
Chapter 3: Mathematically Modeling Probes to Measure Bond Lifetime 21
INTRODUCTION 22
Probe Design 24
Mathematical Model Derivation 25
RESULTS 32
Model Stability 32
Excitation Intensity 33
Lifetime Variation 34
Signal and Signal to Background 37
Results Summary 38
DISCUSSION 39
Simulation Limitations 39
Potential Improvements 41
Lowered Excitation Intensity 42
Long Luminescence Lifetime 43
The Photon Count Conundrum 43
CONCLUSIONS 44
Chapter 4: Tension Probes to Disentangle Force and Work in T-Cell Receptor Triggering 45
INTRODUCTION 46
RESULTS 52
DISCUSSION 69
Hairpin Design 69
Biological Implications 71
CONCLUSIONS 74
METHODS 74
Long Glass Coverslips: Piranha Functionalization. 74
Surface Preparation: Long Slides, APTES. 75
Surface Preparation: Round Coverslips, APTES. 75
TCO conjugation – Round Slides: 75
TCO Conjugation – Long Glass Coverslips: 76
Hairpin Folding: 76
Hairpin Surface Conjugation: 76
Anti-CD3 Functionalization and T-cell Addition: 77
Locking Strands: 77
Methyltetrazine Conjugation: 77
Atto647N Conjugation: 78
Hairpin Melting Analysis: 78
Electrophoretic Gel Separation: 79
Sequences 79
Chapter 5: Conclusions 81
REFERENCES 84
About this Honors Thesis
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