Improving the Accuracy, Versatility, and Partitioning Efficiency of Nonequilibrium Capillary ...

194 Pages·2016·3.83 MB·English

Improving the Accuracy, Versatility, and
Partitioning Efficiency of
Nonequilibrium Capillary Electrophoresis of
Equilibrium Mixtures
Mirzo Kanoatov
A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
GRADUATE PROGRAM IN BIOLOGY
YORK UNIVERSITY
TORONTO, ONTARIO
November 2016

© Mirzo Kanoatov, 2016
ABSTRACT
Studying the affinity and the kinetics of biomolecular interactions is an essential task in
biology and pharmacology. Nonequilibrium capillary electrophoresis of equilibrium mixtures
(NECEEM) is a promising technique that allows the measurement of the equilibrium dissociation
constant (K ), and the rate constant of dissociation (k ), and can also be used as the partitioning
d off
method in selection of aptamers. There are three critical issues that have hindered the wide
adoption of NECEEM by the analytical community: (1) poor resilience of the method against
systematic errors, (2) limited versatility in terms of emulating physiological conditions, and
(3) restricted partitioning efficiency in DNA aptamer selection. The goals of my research project
were (i) to develop strategies for minimizing the systematic errors in NECEEM; (ii) to make
NECEEM compatible with the use of physiological buffers; and (iii) to eliminate the restricted
partitioning efficiency of NECEEM in aptamer selection. To improve the accuracy of NECEEM,
I have developed an approach for simultaneous determination of both the K and the
d
concentration of one of the interacting molecules, which eliminates errors caused by the presence
of improperly folded molecules in samples. Further, I have developed an “algorithmic” approach
for optimization of NECEEM, which takes the interrelation between its experimental parameters
into account and minimizes the systematic error in an objective manner. To make NECEEM
compatible with physiological buffers, I have uncovered the cause behind the poor detectability
of DNA in phosphate buffered saline (PBS), and have created a pressure-assisted modification of
NECEEM that overcomes this problem. Lastly, I have determined that the irregular
electrophoretic migration of DNA in strong electric fields is caused by the ability of DNA to
form unusually stable complexes with its counterions. Based on these findings, I have developed
two independent approaches that increase the efficiency of NECEEM in selection of DNA
aptamers.
ii
to Mom and Dad,
for always placing the needs and aspirations
of their children first

and to Lise,
for being my source of encouragement and motivation,
without whom I would probably take another 10 years to finish

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ACKNOWLEDGEMENTS
First and foremost, I would like to thank my academic supervisor, Professor Sergey N.
Krylov, without whose guidance and support this work would be impossible. Dr. Krylov is a
brilliant scientist, whose passion and dedication to this art will always be my model for imitation.
I was extremely lucky to have such an exceptional teacher, who is genuinely dedicated to the
success of his students. Through his tireless efforts, he is able to provide us with opportunities
that few other students have access to. Thank you for all of the time, energy and resources that
you have dedicated to my development. Most importantly, thank you for giving me your trust,
freedom to pursue my interests, and your genuine care. You have shaped me as a scientist and as
a person, and I carry your gifts with pride.
I would like to express my sincere gratitude to the members of my supervisory committee,
Professors Katalin A. Hudak and Samuel Benchimol, whose wisdom and insight has steered my
ever-changing research project into the right direction. Thank you for your caring, patience and
the invaluable advice that you have given to me over the years. I would also like to thank the
members of my examining committee, Professors Mark Bayfield and Derek J. Wilson, as well as
my external examiner, Professor Andrei Drabovich from University of Toronto, for dedicating
their invaluable time to the evaluation of my work.
In my time spent at the Krylov Lab, I have had the privilege of rubbing shoulders with some
of the brightest and most exceptional people in the history of the Universe. Many have become
my mentors: Sahar Javaherian, Maxim Berezovski, Svetlana Krylova, Michael Musheev,
Alexander Petrov, Victor Galievsky, and Leonid Cherney, all have dedicated countless hours of
their time to share their knowledge and expertise with me. A very special place in my heart will
always be reserved for my fellow grad students: David Wegman, Jiayin Bao, Fletcher Agostino,
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who have been with me from day one, and soon joined by Natalia Obrecht, Roman Yufa, and
Ruchi Liyanage. We grew together, learned from each other, shared in our successes and
failures, saw each other getting married and becoming parents, and downed many liters of beer
while at it. A special mention goes out to Roseanne Evenhuis, whose loving work has made the
lab feel like a second home to us all. I was also extremely fortunate to collaborate with some
exceptional undergraduate students: Coraline Retif, Asela Gamage, Sina Mehrabanfar and An Le
Thi Hoai. The projects that we have worked on together would not have succeeded without your
brilliant ideas, hard work and dedication. All of the members of the Krylov Lab, past and
present, thank you for your friendship and your support.
All of my successes in life I owe to my family. My wife, Lise Geffray, is my rock without
whom I would collapse. Thank you for the love and happiness that you have given me, it has
kept me going through these trying times. Thank you to my parents, Diloro Tursun-Zadeh and
Daler Kanoatov, for your unconditional love, your hard work raising us, and for making me
believe that I can achieve anything. Special recognition goes out to my grandfather, Masud
Tursun-Zadeh, and my uncles, Nodir and Irage Tursun-Zadeh, who have instilled in me the love
of science and technology from a young age. All of the members of my large family, thank you
for your love, kindness and support. I would not be where I am today without you (quite
literally).
I would also like to express my sincere appreciation to Biology Graduate Assistants, Adrienne
Dome and Cristalina Del Biondo, whose tireless efforts made the lives of countless graduate
students much easier. My endless gratitude goes out to York University, for being my second
home for over a decade. I would also like to thank Natural Sciences and Engineering Research
Council of Canada and the Government of Ontario for financial support.
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TABLE OF CONTENTS
ABSTRACT .................................................................................................................................... ii
DEDICATION ............................................................................................................................... iii
ACKNOWLEDGEMENTS ........................................................................................................... iv
TABLE OF CONTENTS ............................................................................................................... vi
LIST OF TABLES ...........................................................................................................................x
LIST OF FIGURES ....................................................................................................................... xi
LIST OF ABBREVIATIONS ...................................................................................................... xiii
LIST OF SYMBOLS .....................................................................................................................xv
LIST OF EQUATIONS ............................................................................................................... xix
Chapter 1. Introduction

1.1. STUDYING BIOMOLECULAR INTERACTIONS ...............................................................1
1.1.1. Characteristics of biomolecular interactions ................................................. 1
1.1.2. Analytical performance parameters of methods for studying BMIs ................ 2
1.1.3. Importance of measuring kinetics of BMIs ................................................... 4
1.2. METHODS FOR STUDYING THE KINETICS OF BMIS ....................................................6
1.2.1. Surface plasmon resonance spectroscopy and bio-layer interferometry .......... 6
1.2.2. Stopped-flow techniques .............................................................................. 7
1.2.3. Fluorescence correlation spectroscopy .......................................................... 8
1.2.4. Kinetic capillary electrophoresis .................................................................. 9
1.2.4.1. Basic principles of CE.......................................................................................................... 9
1.2.4.2. Concept of KCE ................................................................................................................. 11
1.3. NECEEM – THE PRINCIPLE KCE METHOD ....................................................................13
1.3.1. Concept of NECEEM ................................................................................. 13
1.3.2. History of NECEEM development and application ...................................... 17
1.3.3. Technological limitations of NECEEM and project goals ............................ 21
Chapter 2. Using NECEEM for Simultaneous
Determination of Concentration and Equilibrium
Constant

2.1. ERRORS DUE TO UNCERTAINTY IN TARGET CONCENTRATION ...........................25
2.2. EXPERIMENTAL SECTION ................................................................................................27
2.2.1. Materials ................................................................................................... 27
2.2.2. Capillary electrophoresis ............................................................................ 27
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2.2.3. NECEEM ................................................................................................... 29
2.3. RESULTS AND DISCUSSION .............................................................................................30
2.3.1. Mathematical solution for simultaneous determination of K and [T] ......... 30
d 0
2.3.2. Considerations for avoiding systematic errors ............................................ 30
2.3.3. In silico modeling and validation. .............................................................. 34
2.3.4. Experimental determination of K and [T] ................................................. 40
d 0
2.4. CONCLUSIONS.....................................................................................................................43
Chapter 3. Systematic Approach to Optimization of
NECEEM Experimental Conditions

3.1. INTRODUCTION TO NECEEM OPTIMIZATION .............................................................44
3.2. EXPERIMENTAL SECTION ................................................................................................46
3.2.1. Materials ................................................................................................... 46
3.2.2. NECEEM ................................................................................................... 46
3.3. RESULTS AND DISCUSSION .............................................................................................48
3.3.1. Defining the Parameters and the Variables ................................................. 48
3.3.2. Phase 1: Experiment validation .................................................................. 50
3.3.3. Phase 2: Recommendations for adjustment of experimental conditions ....... 56
3.3.4. Modifications for preparative applications .................................................. 62
3.3.5. Recommended conditions for an initial experiment ..................................... 63
3.3.6. Software implementation ............................................................................ 64
3.3.7. Practical application ................................................................................... 66
3.4. CONCLUSIONS.....................................................................................................................68
Chapter 4. NECEEM Analysis of DNA in Physiological
Conditions

4.1. IMPORTANCE OF MEASUREMENTS IN PHYSIOLOGICAL CONDITIONS ...............69
4.2. EXPERIMENTAL SECTION ................................................................................................71
4.2.1. Materials ................................................................................................... 71
4.2.2. Capillary electrophoresis ............................................................................ 71
4.2.3. NECEEM ................................................................................................... 72
4.2.4. Mobility measurements .............................................................................. 74
4.2.5. Peak broadening measurements .................................................................. 75
4.2.6. Characterization of pressure-assisted NECEEM .......................................... 75
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4.3. RESULTS AND DISCUSSION .............................................................................................75
4.3.1. Issue with DNA detectability in PBS-NECEEM .......................................... 75
4.3.2. Fluorescence intensity ................................................................................ 79
4.3.3. Analyte velocity ......................................................................................... 81
4.3.4. Peak broadening ......................................................................................... 86
4.3.5. Pressure-facilitated NECEEM .................................................................... 90
4.4. CONCLUSIONS.....................................................................................................................94
Chapter 5. Non-Uniform Velocity of Homogeneous DNA
in a Uniform Electric Field

5.1. REDUCED PARTITIONING EFFICIENCY OF NECEEM FOR DNA MOLECULES .....96
5.2. EXPERIMENTAL SECTION ................................................................................................98
5.2.1. Materials ................................................................................................... 98
5.2.2. Capillary electrophoresis .......................................................................... 100
5.2.3. Quantitative polymerase chain reaction .................................................... 101
5.2.4. DNA dialysis ........................................................................................... 101
5.2.5. Solubility of DNA aggregates. ................................................................. 102
5.2.6. DNA integrity .......................................................................................... 103
5.3. RESULTS AND DISCUSSION ...........................................................................................104
5.3.1. Non-uniform velocity of DNA .................................................................. 104
5.3.2. Possible causes for non-uniform velocity of DNA .................................... 106
5.3.3. Interactions of DNA with its counterions .................................................. 108
5.3.4. DNA precipitation by electro-dialysis ...................................................... 115
5.3.5. Improving partitioning efficiency of NECEEM without desalting ............. 120
5.4. CONCLUSIONS...................................................................................................................123
Chapter 6. Concluding Statements

6.1. OUTCOMES AND IMMEDIATE APPLICATIONS ..........................................................125
6.2. LIMITATIONS AND FUTURE DIRECTIONS ..................................................................127
LIST OF PUBLICATIONS .........................................................................................................131
REFERENCES ............................................................................................................................132
APPENDIX A. MATHEMATICAL DERIVATIONS AND PROOFS ..................................... 143
A.1. Relationship between [L] and error in measuring of K ............................ 143
0 d
A.2. Proof for Optimum Relation between K and [T] ..................................... 143
d 0
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APPENDIX B. EXTRACTING KINETICS FROM AFFINITY CE DATA ............................. 146
B.1. Summary .................................................................................................. 146
B.2. Introduction ............................................................................................. 146
B.3. Results and discussion .............................................................................. 149
B.4. Concluding remarks .................................................................................. 169
B.5. Appendix references ................................................................................. 169

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LIST OF TABLES
Table 1.1. Comparison of methods for studying BMI kinetics ....................................................... 5
Table 3.1. Required information about the limitations of the CE instrument ............................... 57
Table 3.2. Recommendations for experimental conditions for a study-initiating experiment ...... 64

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