Combining Single Molecule Fluorescence and Force Experiments: Multiplexed Assays Público

Abstract

Single molecule fluorescence resonant energy transfer allows for following conformational changes of biomolecules in the sub-nanometer range with millisecond time resolution. The technique has been used to follow conformational dynamics of DNA and its interactions with DNA binding proteins in vitro. Inside the cell, DNA is physically constrained, so binding and translocation of proteins along the DNA result in application of torques and forces that may have an effect on its thermodynamic stability. It is important to understand the effect of physical constraints on the DNA because local conformation and flexibility of DNA have an effect in its interaction with proteins. Proof of principle type of experiments, in which DNA manipulation is combined with single molecule fluorescence detection, have been reported. In practice, the experiments are highly challenging from a technical point of view, limiting their scope of applications. In this work we improve and develop a series of techniques that allow generation of long DNA substrates (11 kbp in this case) with asymmetric ends for high yield/high efficiency selective labeling. Long DNA substrates with asymmetric ends were generated by over expression of plasmids in E. coli. Fragments were purified with high yields and adaptor DNA sequences attached selectively to the long DNA fragments using a low temperature ligation reaction compatible with long term stability of fluorescent dyes. To demonstrate high efficiency double-labeling of the long DNA fragments we specifically immobilized DNA on a microscope slide surface with a biotin/streptavidin linkage. The DNA free end was attached to antidigoxigenin coated polystyrene beads. Application of buffer at several flow rates was used to determine the yield of doubly-labeled substrates and to investigate the range of forces that can be applied with this scheme. Our results show that we can immobilize doubly-labeled substrates at high densities compatible with single molecule fluorescence observation and a large range of forces.

Table of Contents

Table of Contents
1 Introduction....................................................................................1 1.1 Background..................................................................................1

1.2 The Ultimate Goal: Combining smFRET and Force Experiments................6

1.3 Hydrodynamic and Electrophoretic Stretching.....................................8

1.4 Atomic Force Microscopy (AFM)......................................................11 1.5 Optical Tweezers (OT)..................................................................13 1.6 Magnetic Tweezers (MT)...............................................................16

1.7 Plasmid cloning for long DNA strand amplification................................18

1.8 Adaptor DNA end-labeling..............................................................21 2 Results.........................................................................................23 3 Conclusion and Future Applications....................................................32 Bibliography......................................................................................36

Appendix A: List of Tables...................................................................39

Appendix B: Materials and Methods.......................................................41

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List of Figures -

Figure 1: WLC Model Theoretical Force-Extension Curve.............................5

Figure 2: Combining smFRET and Force Manipulation Experiments..................8

Figure 3: Hydrodynamic Stretching........................................................10

Figure 4: Electrophoretic Stretching.......................................................11

Figure 5: Atomic Force Microscopy.........................................................13

Figure 6: Optical Tweezers...................................................................16

Figure 7: Magnetic Tweezers................................................................18

Figure 8: Cloning and Transformation Schematic Illustration........................21

Figure 9: Adaptor DNA Ligation Schematic Illustration................................22

Figure 10: EtBr Stained 1% Agarose Gel..................................................25

Figure 11: Slide/Coverslip Construction...................................................26

Figure 12: Total Internal Reflection Microscopy.........................................27

Figure 13: Large Mean Density of Specifically Bound DNA Molecules..............29

Figure 14: Flow Rate vs. Extension Curve................................................30

Figure 15: Antidigoxigenin Polystyrene Beads............................................31

Figure 16: Two Common Flow Assay Configurations...................................33

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List of Tables -

Table 1: Oligonucleotides Used..............................................................39

Table 2: Restriction Enzymes.................................................................40

Table 3: Adaptor DNA..........................................................................40

About this Honors Thesis

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School	Emory College
Department	Physics
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biophysics, General
Palabra Clave	single molecule fluorescence multiplex assay hydrodynamic DNA extension optical tweezers smFRET magnetic tweezers flow force manipulation
Committee Chair / Thesis Advisor	Rasnik, Ivan, Emory University
Committee Members	Borthwick, David, Emory University Brody, Jed, Emory University

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