Ultrasensitive Tension Sensors with Catalytic Amplification Reactions: From Fundamental Development to Clinical Applications Público
Duan, Yuxin (Summer 2022)
Published
Abstract
Cells transmit piconewton forces to their receptors to mediate essential biological processes such as coagulation and immune recognition. A major challenge is that cell-generated forces are infrequent, transient, and difficult to detect. In this dissertation, we introduce the development and application of ultrasensitive tension sensors using a number of mechanically triggered catalytic amplification reactions. These ultrasensitive tension sensors and assays, once optimized, allow one to detect cell forces without the need for microscopy instrumentation. Chapter 1 of this dissertation introduces the current state of mechanobiology, as well as methods to detect cellular forces and catalytic amplification reactions. Chapter 2 describes the development and application of the mechanically triggered hybridization chain reaction (mechano-HCR). Building on the work, Chapter 3 summarizes the mechano-Cas12a assisted tension sensor which offers greater amplification and hence improves sensitivity compared to mechano-HCR. We show that both of these catalytic amplification assays provide tools to detect receptor mediated forces generated by fibroblasts, platelets, and T cells in a high-throughput manner. We further show that cellular forces may be used as a diagnostic marker for predicting post-surgery bleeding risk and for personalized tailoring of anti-coagulant drugs. Finally, Chapter 5 summarizes the work in this dissertation and discusses its future outlook.
Table of Contents
Chapter 1. Introduction of Mechanotransduction, Receptor Force Detection Methods and Catalytic Amplification Reactions. 1
1.1 Introduction of Mechanobiology and Receptor Mediated Forces 2
1.2 Mechanotransduction Mediated by Integrin Receptors 3
1.2.1 General background of integrin mediated mechanotransduction 3
1.2.2 Platelet integrins and integrin tension in platelet function 5
1.3 Methods in Cellular Force Detection 9
1.3.1 DNA based tension probes 10
1.3.1.1 Reversible DNA tension probes 11
1.3.1.2 Irreversible DNA tension probes 12
1.3.1.3 Other Development and Limitations of the DNA Tension Probes 13
1.4 Catalytic Amplification Reactions 14
1.4.1 Hybridization chain reaction 16
1.4.2 CRISPR-Cas12a nuclease reaction 17
1.5 Aim and Scope of the Dissertation 20
1.6 References 22
Chapter 2. Development of Mechanically-triggered Hybridization Chain Reaction 30
2.1 Abstract 31
2.2 Introduction 31
2.3 Results and Discussion 37
2.3.1 Validation and characterization of HCR on surface 37
2.3.2 Mapping Live-cell tension signal with mechano-HCR 39
2.3.3 Characterization of mechano-HCR with cells and super-resolved HCR 42
2.3.4 Rapid detection of cellular tension with mechano-HCR and plate reader 43
2.3.5 Measuring dose-response function of cell-mechanics modulating molecules with mechano-HCR 44
2.3.6 Drug screening of inhibitors for mouse platelet mechanics with mechano-HCR 46
2.4 Conclusion 50
2.5 Materials and Methods 52
2.5.1 Chemicals and oligonucleotides 52
2.5.2 Instruments 54
2.5.3 Surface preparation method 54
2.5.4 DNA hybridization 55
2.5.5 Oligo dye/ligand coupling and purification 56
2.5.6 Solution based hybridization chain reaction (HCR) and agarose gel electrophoresis 57
2.5.7 Mouse platelet handling 58
2.5.8 Cell culture 58
2.5.9 Mechanically-triggered hybridization chain reaction 59
2.5.10 Dose-dependent inhibition of integrin mediated tension 59
2.5.11 Microscopy imaging 60
2.5.12 Determination of DNA surface density 61
2.6 Acknowledgement 62
2.7 Appendix 63
2.8 References 84
Chapter 3. Development of Mechano-Cas12a Assisted Tension Sensor 92
3.1 Abstract 93
3.2 Introduction 93
3.3 Results 96
3.3.1 Design and optimization of MCATS 96
3.3.2 Rapid, robust, ultrasensitive detection of cellular tension with MCATS 100
3.3.3 High-throughput determination of platelet inhibitors’ influence on platelet tension 104
3.3.4 Platelet tension predicts transfusion need in CPB patients 108
3.4 Discussion 112
3.5 Conclusion 115
3.6 Materials and Methods 116
3.6.1 Chemicals and oligonucleotides 116
3.6.2 Instruments 118
3.6.3 Surface preparation method 118
3.6.4 DNA hybridization and gRNA/Cas12a binding 119
3.6.5 Oligo dye/ligand coupling and purification 119
3.6.6 Solution based Cas12a amplification and plate reader readout 120
3.6.7 Human platelet handling and Ethics agreement 121
3.6.8 Cell culture 121
3.6.9 Mechano-Cas12a assisted tension sensor 122
3.6.10 Dose-dependent inhibition of receptor mediated tension 122
3.6.11 Microscopy imaging 123
3.6.12 Statistics and reproducibility 123
3.7 Acknowledgement 124
3.8 Appendix 124
3.9 References 143
Chapter 4. Investigate T cell Mechanics with Reversible Tension Probe MCATS (RT-MCATS) 148
4.1 Abstract 149
4.2 Introduction 149
4.3 Results and Discussion 152
4.3.1 Design of MCATS with reversible DNA tension probe 152
4.3.2 Optimization of RT-MCATS design with strain-free strategy and rapid detection of TCR forces with altered peptide ligands. 155
4.3.3 Rapid detection of mechanics modulating drug’s effect on T cell 158
4.4 Conclusion 160
4.5 Materials and Methods 161
4.5.1 Chemicals and oligonucleotides 161
4.5.2 Instruments 163
4.5.3 Surface preparation method 164
4.5.4 DNA hybridization and gRNA/Cas12a binding 165
4.5.5 Oligo dye/ligand coupling and purification 165
4.5.6 T cell handling 166
4.5.7 MCATS assay with reversible tension probes. 166
4.5.8 Dose-dependent inhibition of receptor mediated tension 167
4.5.9 Microscopy imaging 167
4.5.10 Statistics and reproducibility 168
4.6 Acknowledgement 168
4.7 Appendix 169
4.8 References 173
Chapter 5. Summary and Perspective 175
5.1 Summary 176
5.2 Perspective 179
5.2.1 Further development of mechano-HCR and MCATS 179
5.2.1.1 Optimization of assay running time and sensitivity 179
5.2.1.2 Increasing the versatility of the assays 181
5.2.1.3 Standardizing and simplifying the experimental procedure 181
5.2.2 Future applications for ultrasensitive tension sensors 182
5.2.2.1 Detection of platelets mechanics and clinical applications 182
5.2.2.2 Tension history profile with Exchange HCR 183
5.2.2.3 Mechanical proofreading of interactions of target molecules with flow induced MCATS 184
5.2.2.3 Other applications 191
5.3 Other Contributions 191
5.4 Closing Remarks 193
5.5 References 193
About this Dissertation
| School | |
|---|---|
| Department | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Palavra-chave | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Ultrasensitive Tension Sensors with Catalytic Amplification Reactions: From Fundamental Development to Clinical Applications () | 2022-08-07 15:46:33 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Movie 2.1: Real time mechano-HCR amplification in cell experiments. (Raw timelapse of fibroblasts showing the mechano-HCR with hairpins in 240 minutes time window. Tension signal in TIRF quickly increased during the first 2h. Scale bar (in white) = 12 µm) | 2022-08-05 16:21:02 -0400 |
|
|
|
Movie 2.2: Z stack images of mechano-HCR signal with a confocal microscope. (Confocal images of Z stack show that mechano-HCR signal mainly locate at the cell-surface interaction. Scale bar (in white) = 8 µm.) | 2022-08-05 16:21:13 -0400 |
|
|
|
Movie 5.1: Flow induced shearing of anti-dig beads with dig DNA strands. (Raw timelapse of the RICM channels showing the beads are sheared at 5s.) | 2022-08-05 16:21:25 -0400 |
|