Development of methods for mechanical manipulation of biomolecules and super-resolution measurement of cellular forces Restricted; Files Only
Su, Hanquan (Summer 2020)
Abstract
In general, our understanding of how mechanics influences biology remains at its infancy, and especially when compared to our understanding of how chemical signals influence biological systems. Techniques to apply and measure forces at the molecular scale are central to address this challenge. One widely used family of techniques is single molecule force spectroscopy (SMFS), which employs a dedicated instrument to apply forces and measure the extension of individual molecules. However, because of its serial nature, SMFS remains cumbersome and suffers from limited throughput hindering its utility. In this thesis, we develop polymer force clamp (PFC) particles that can apply forces to biomolecules in a massively parallelized fashion, thus solving the throughput problem plaguing SMFS since its inception. Complementing this effort, we also develop new probes to map the molecular forces generated by cells with nanoscale spatial resolution. Together, these tools help reveal the role of mechanical forces in biology.
Chapter 1 provides an overview of the conventional and state-of-the-art SMFS manipulation methods and their limitations in terms of resolution, throughput and speed.
Chapter 2 details the development of the light-responsive PFC for pN force manipulation of DNA hairpins. PFCs are comprised of a gold nanorod coated with a responsive polymer shell and collapses to generate mechanical work upon illumination.
Chapter 3 characterizes the rate of PFC volume phase transition using time-resolved spectroscopy. The work demonstrates the fastest recorded response time of a hydrogel material with nanoscale heating.
Chapter 4 develops methods for time-resolved fluorescence analysis of mechanical unfolding of DNA hairpins. We demonstrate parallel mechanical unfolding of an ensemble of DNA hairpins at microsecond time scales.
Chapter 5 reports the application of a super resolution imaging technique (DNA-PAINT) to map the mechanical unfolding of DNA tension probes due to the pN forces generated by cells. The method provides the highest resolution maps of cellular forces reported to date.
Finally, Chapter 6 summarizes the work in this thesis and discusses its future outlook. We believe that this thesis will promote the evolution of biophysical tools that impact the fields of sensing, diagnosis, and mechanobiology.
Table of Contents
Chapter 1. Introduction of methods to study biomolecular and cellular forces ............................................ 1
1.1 Introduction ......................................................................................................................................... 2
1.1.1 Examples of conventional force manipulation methods .............................................................. 2
1.1.2 Limitations of conventional force manipulation methods............................................................ 5
1.1.3 An overview of methods to study cellular forces ......................................................................... 8
1.2 Development of parallel force manipulation techniques .................................................................. 10
1.2.1 Parallel AFM .............................................................................................................................. 10
1.2.2 High-throughput optical trapping: from 2D surface to 3D bulk solution .................................. 12
1.2.3 Acoustic tweezer ........................................................................................................................ 13
1.2.4 Centrifuge force microscope ...................................................................................................... 14
1.2.5 DNA-based molecular force spectroscopy ................................................................................. 15
1.3 Development of high-speed force manipulation techniques ............................................................. 17
1.3.1 An overview of the gap between experiment and MD simulation ............................................. 17
1.3.2 High-speed AFM........................................................................................................................ 18
1.4 Stimulus-responsive polymer and its application on mechanical actuation. ..................................... 19
1.4.1 An introduction of stimulus-responsive polymer ....................................................................... 19
1.4.2 Optomechanical actuator and its application on cellular mechanics .......................................... 20
1.4.3 Stimulus-responsive polymer particle for force manipulation on biomolecules ........................ 22
1.5 High resolution imaging of cellular forces and force-bearing cellular structures ............................. 23
1.5.1 Diffraction-limited measurement of cellular forces ................................................................... 23
1.5.2 Super resolution imaging of nanoscale force-bearing cellular structures .................................. 24
1.6 Aim and scope of the dissertation ..................................................................................................... 25
1.7 References ......................................................................................................................................... 26
Chapter 2 Development of bottom-up synthesized particle force clamp for mechanical unfolding of biomolecules ............................................................................................................................................... 35
2.1 Abstract ............................................................................................................................................. 36
2.2 Introduction ....................................................................................................................................... 36
2.3 Results ............................................................................................................................................... 38
2.3.1 Synthesis and characterization of PFC ....................................................................................... 38
2.3.2 Mechanical clamping of DNA hairpin ....................................................................................... 40
2.3.3 PFC delivers dynamic and tunable pN forces ............................................................................ 43
2.3.4 Calibration of the force generated by PFC ................................................................................. 44
2.3.5 Single molecule unfolding using PFC ....................................................................................... 46
2.4 Discussion and conclusion ................................................................................................................ 47
2.5 Materials and methods ...................................................................................................................... 48
2.5.1 Materials .................................................................................................................................... 48
2.5.2 Synthesis of N, N’-bis(acryloyl)cystamine stabilized AuNRs ................................................... 49
2.5.3 Synthesis of 13 nm AuNPs ........................................................................................................ 49
2.5.4 Synthesis of Polymer Force Clamp (PFC) particles .................................................................. 50
2.5.5 Transmission electron microscopy (TEM) ................................................................................. 50
2.5.6 DNA labeling ............................................................................................................................. 51
2.5.7 Surface modification .................................................................................................................. 51
2.5.8 Synthesis of AuNP-DNA to determine the stoichiometry between AuNPs and DNA hairpins 52
2.5.9 Fluorescein labeling of PFC ....................................................................................................... 52
2.5.10 Azide-alkyne cycloaddition click reaction ............................................................................... 53
2.5.11 UGA-firefly galvo system integrated fluorescence microscopy .............................................. 53
2.5.12 DNA unfolding imaging .......................................................................................................... 53
2.5.13 Single molecule imaging .......................................................................................................... 54
2.5.14 Estimation of tension tolerance (Ttol) of TGT .......................................................................... 55
2.5.15 Determination of the change in fluorescence intensity (I) of each particle ........................... 55
2.5.16 Integration of fluorescence intensity ........................................................................................ 55
2.5.17 Determination of the temperature gradient around PFC by finite element analysis ................ 56
2.6 References ......................................................................................................................................... 56
2.7 Appendix ........................................................................................................................................... 61
Chapter 3: Characterization of the volume phase transition rate of the PNIPMAm-based polymer particle .................................................................................................................................................................... 83
3.1 Abstract ............................................................................................................................................. 84
3.2 Introduction ....................................................................................................................................... 84
3.3 Results ............................................................................................................................................... 87
3.3.1. Spectroscopic characterization of equilibrium volume-phase transition .................................. 87
3.3.2. Pump-probe measurement of phase transition dynamics .......................................................... 91
3.3.3. Finite-element simulation of heat transfer dynamics ................................................................ 95
3.4 Discussion and conclusion ................................................................................................................ 98
3.5 Materials and methods .................................................................................................................... 102
3.5.1 Materials .................................................................................................................................. 102
3.5.2 Synthesis of polymer force clamp (PFC) particles .................................................................. 102
3.5.3 PFC particle and AuNR characterization ................................................................................. 104
3.5.4 Temperature-dependent steady-state FTIR measurement ........................................................ 104
3.5.5 Local T-jump transient IR and near-IR absorption spectroscopy measurement ...................... 104
3.5.6 Global T-jump time-resolved IR measurement ........................................................................ 105
3.5.7 Spectral data analysis ............................................................................................................... 106
3.5.8 Finite element analysis and heat transfer simulations of PFC particle .................................... 106
3.5.9. Principal component analysis (PCA) of temperature-dependent steady-state FTIR spectra .. 107
3.6 References ....................................................................................................................................... 109
3.7 Appendix ......................................................................................................................................... 117
Chapter 4: Time-resolved chemical analysis of biomolecule unfolding with micro-polymer force clamp .................................................................................................................................................................. 131
4.1 Abstract ........................................................................................................................................... 132
4.2 Introduction ..................................................................................................................................... 132
4.3 Results ............................................................................................................................................. 134
4.3.1 Synthesis and characterization of OPFC assembly .................................................................. 134
4.3.2 DNA hairpin parallel unfolding using micro-polymer force clamp in bulk solution ............... 137
4.3.3 Time-resolved force-jump fluorescence measurement of DNA unfolding .............................. 140
4.4 Discussion and conclusion .............................................................................................................. 143
4.5 Materials and methods .................................................................................................................... 143
4.5.1 Materials. ................................................................................................................................. 143
4.5.2 Synthesis of micro-polymer force clamp. ................................................................................ 144
4.5.3 Transmission electron microscopy (TEM) ............................................................................... 145
4.5.4 DNA labeling. .......................................................................................................................... 145
4.5.5 Equilibrium fluorescence measurement ................................................................................... 146
4.5.6 Time-resolved T-jump fluorescence spectroscopy .................................................................. 146
4.5.7 Data acquisition and analysis of force ..................................................................................... 147
4.6 References ....................................................................................................................................... 149
4.7 Appendix ......................................................................................................................................... 151
Chapter 5: Super resolution measurement of pN cellular traction forces ................................................. 161
5.1 Abstract ........................................................................................................................................... 162
5.2 Introduction ..................................................................................................................................... 162
5.3 Results ............................................................................................................................................. 163
5.3.1 DNA-PAINT on conventional DNA tension probes ............................................................... 163
5.3.2 Strain-free tension-PAINT of cellular mechanical force ......................................................... 163
5.3.3 Filtration of background single molecule fluorescence signal ................................................. 165
5.3.4 Exchange PAINT imaging of cellular mechanics related protein. ........................................... 165
5.3.5 Accumulated tension PAINT imaging of cellular mechanical force. ...................................... 167
5.4 Discussion and conclusion .............................................................................................................. 168
5.5 Materials and methods .................................................................................................................... 174
5.5.1 Materials .................................................................................................................................. 174
5.5.2 Surface preparation .................................................................................................................. 174
5.5.3 Imaging chamber assembly ...................................................................................................... 175
5.5.4 DNA hybridization ................................................................................................................... 175
5.5.5 Platelet handling ....................................................................................................................... 175
5.5.6 Cell culture ............................................................................................................................... 176
5.5.7 Single molecule localization imaging for Tension-PAINT...................................................... 176
5.5.8 Exchange-tPAINT .................................................................................................................... 177
5.5.9 Image processing...................................................................................................................... 177
5.5.10 Synthesis ................................................................................................................................ 179
5.5.11 Origami design and synthesis ................................................................................................ 180
5.5.12 Determination of DNA surface density.................................................................................. 181
5.5.13 Statistics ................................................................................................................................. 182
5.5.14 Probe design considerations ................................................................................................... 182
5.5.15 Imager binding kinetics to mechanically strained docking sites ............................................ 186
5.5.16 Experimental considerations for tPAINT measurements ....................................................... 193
5.6 References ....................................................................................................................................... 197
5.7 Appendix ......................................................................................................................................... 204
Chapter 6: Summary and Perspective ....................................................................................................... 240
6.1 Summary ......................................................................................................................................... 241
6.2 Perspective ...................................................................................................................................... 242
6.2.1 Extremely high speed force spectroscopy ................................................................................ 243
6.2.2 Mechanical regulation on chemical reactions in bulk solution ................................................ 243
6.2.3. In-vivo force manipulation ...................................................................................................... 244
6.2.4 Application of high-throughput force spectroscopy on diagnosis ........................................... 245
6.3 Other contributions ......................................................................................................................... 246
6.4 References ....................................................................................................................................... 248
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