Developing and Understanding Nucleic Acid Tension Sensors to Dissect Cellular Mechanosensing Restricted; Files Only

Shinde, Pushkar (Spring 2022)

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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


RESULTS          9

+ Spacer Hairpins        11

-  Spacer Hairpins        13


Effect of Structure on Mechanics       14

Structure and Photophysics   16


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


Simulation Limitations            39

Potential Improvements         41

Lowered Excitation Intensity  42

Long Luminescence Lifetime  43

The Photon Count Conundrum          43


Chapter 4: Tension Probes to Disentangle Force and Work in T-Cell Receptor Triggering   45

INTRODUCTION          46

RESULTS          52


Hairpin Design 69

Biological Implications            71


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


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