Determining the Exonuclease Velocity of DNA Polymerase at
Different Forces with Acoustic Force Spectroscopy
Benno Peters
July 2019
Report Bachelor Project Physics and Astronomy
Size Project
15 EC
Student Number
10735623
Conducted Between
04/2019 - 07/2019
Name Institute
VU, Vrije Universiteit
UvA, Universiteit van Amsterdam
Name of Faculty
Faculty of Science
Date of Submission
6 July 2019
Supervisors
Kiki H. Taris, MSc.
Prof. dr. ir. Gijs J.L. Wuite
Abstract
This thesis is the first step in a much bigger process for finding and improving cancer cures. Cancer is in most western countries the leading cause of death before the age of 70 years old with almost 20 million new cancer cases worldwide every year, which is expected to double in the next 20+ years. With a projected total health care expenditure cost for cancer of $180-200 billion US dollars in 2022, it is vitally important that solutions are found for those problems.
The first step in this process, which is the aim of this research, is to find out whether it is possible with Acoustic Force Spectroscopy (AFS) to research DNA replication. This is done by determining the exonuclease velocity of DNA polymerase (DNAp), the enzyme that makes DNA replication happen, at multiple constant forces. AFS is a single-molecule technique that allows hundreds of molecules being measured and tracked within nanometer and millisecond accuracy. However, the velocity of DNAp at only 20pN is acquired. Then, this velocity is compared to the established DNAp activity values known from optical tweezer experiments from Hoekstra et al. (2014), to check whether AFS is a viable method to research molecules as DNAp.
If this is possible, step 2 would be to find out whether DNA replication, but also translation and transcription can be researched in more detail. Next, step three would be trying to find and develop better cancer prohibiting medication that binds to DNA. As a preliminary conclusion, the acquired DNAp data with AFS is in the same range as the data acquired with optical tweezers. However, many measurements did not yield high quality data. This could be the case because of ineffective passivation of BSA and pluronics, which allows for beads to bind aspecifically to the surface. Also, the anti-dig might not bind optimally with the surface. Furthermore, the DNA
concentration could be too high, which results in double tethered DNA strands to the same bead. For a definite conclusion, more data is needed at different forces.
Dutch Abstract (Nederlandse Samenvatting)
Kanker is de meest voorkomende doodsoorzaak in de meeste westerse landen, ook in Nederland is dit het geval. Wereldwijd zijn er bijna 20 miljoen nieuwe kankergevallen per jaar, het is uitgerekend dat dit in de komende 20+ jaar zal gaan verdubbelen. Ook lopen de kanker kosten voor medicatie en kankerbehandelingen enorm hoog op, met een verwachte totale kosten voor kanker van $200 miljard US dollars in 2022. Dit vraagt met spoed naar drastische oplossingen.Dit onderzoeksproject is het begin van een veel groter proces voor de mogelijke ontwikkeling van betere en nieuwe kanker medicatie. De eerste stap in dit proces is de focus van dit onderzoek. In dit onderzoek wordt gekeken of DNA-replicatie onderzocht kan worden met Acoustic Force
Spectroscopy (AFS). AFS is een onderzoekstechniek waarbij een akoestische golf wordt gegenereerd die een kracht uitoefent op honderden moleculen. Deze moleculen kunnen met AFS individueel in detail onderzocht worden met nanometer en milliseconde precisie. Als AFS daadwerkelijk een juiste methode is om dit te onderzoeken, kan dit erg helpen het onderzoeksproces naar kanker medicatie te versnellen omdat met AFS honderden moleculen tegelijkertijd kunnen worden onderzocht terwijl met de tegenwoordig meest gebruikte technieken slechts één molecuul tegelijk in detail kan worden onderzocht. Om erachter te komen of AFS inderdaad een goede methode is om DNA-replicatie te onderzoeken wordt in dit onderzoek de activiteit van het enzym DNA polymerase (DNAp) bepaald bij verschilde constante krachten. Echter alleen hoge kwaliteit data is gevonden bij 20 pico newton.
Al het leven in het universum is afhankelijk van juiste DNA-replicatie. Als DNA-replicatie fout gaat kan er genetische informatie verloren gaan en ook is het mogelijk dat dit leidt tot DNA-schade. DNAp is het enzym dat DNA-replicatie mogelijk maakt op een zo goed mogelijke manier. DNAp kan van één streng DNA een dubbele streng DNA maken door als het ware de basenparen aan elkaar te rijgen, polymerisatie genoemd. Ook werkt DNAp de andere kant op, dit wordt exonulease genoemd, dit is het proces wat onderzocht is in dit onderzoeksproject. Met de activiteit wordt hier dan ook bedoeld, de snelheid waarmee DNAp de dubbele DNA streng uit elkaar haalt. Deze activiteit wordt onderzocht met AFS en vergeleken met de bekende literatuur waarde van Hoekstra et al. (2014) om te kijken of inderdaad met AFS de juiste waardes worden gevonden.
Als dit het geval is, is de tweede stap om te kijken of DNA-replicatie, maar ook translatie en transcriptie in meer detail onderzocht kunnen worden. Vervolgens is stap drie het vinden en
ontwikkelen van betere kanker medicatie dat aan DNA bindt, dat potentieel miljoenen levens zou kunnen redden. De voorlopige conclusie is dat de gevonden activiteit van het DNAp in
overeenstemming is met de vooraf bekende literatuur waarde. Echter veel metingen resulteerden niet in hoge kwaliteit data. Dit komt hoogstwaarschijnlijk door een aantal belangrijke factoren zoals ineffectieve passivatie van BSA en pluronics. Ook kan het komen doordat de anti-dig niet optimaal bindt met de oppervlakte. Verder kan een te hoge DNA concentratie ervoor zorgen dat de meting drastisch verstoord wordt omdat meerdere DNA strengen dan aan dezelfde bead kunnen gaan binden. Voor een definitieve conclusie is het belangrijk dat er meer data wordt verzameld bij verschillende krachten.
Acknowledgements
My deepest appreciation goes to Kiki H. Taris for his daily guidance and expertise throughout this bachelor project. With Kiki’s guidance I have developed a larger understanding and passion for biophysics that is invaluable for me. I would like to offer my special thanks to Iddo Heller for
arranging this research project for me. Furthermore, I am particularly grateful to Erwin J.G. Peterman and Gijs J.L. Wuite for giving me the opportunity to do research within their research group. Also, I want to thank the rest of the research group for their support. Moreover, I want to thank the Vrije Universiteit and the Universiteit van Amsterdam for allowing me to do research in their laboratories and for the use of their research equipment.
Contents
1. Introduction 5
1.1 Motivation and Relevance 6
1.2 Acoustic Force Spectroscopy 9
1.3 T7 DNA-Polymerase 10
2. Methods 11
2.1 Experimental Setup 11
2.2 Flow Cell Preparation Method 13
2.3 Method of Bead and DNAp Measurement 14
2.4 Method of Calibration: Conversion Factor from Voltage to Force 15
3. Results and Discussion Analysis 18
4. Conclusion 22
1. Introduction
In the last 10 years, a revolution has begun in the field of science. Especially biosciences and
biotechnology have evolved to a new level, with a greater level of detail, i.e. higher resolution, which could unravel many of the biggest unanswered questions in biological sciences. Imagine, if one could observe biochemical reactions, track and measure particular single macromolecules in a living cell within milliseconds, and track their position within nanometers accuracy. Answers could be found to important questions as: “How is chromosomal DNA replicated?”, or “How does a particular gene get turned on and off, and how do transcription and translation processes occur in real time?”, but also “How do DNA repair mechanisms restore the integrity of incorrectly synthesized or damaged DNA?” Those questions might finally be answered within this revolution. (Xie et al., 2008)
The way molecular properties are measured has changed dramatically. Traditionally biological research is focused on an enormous amount of molecules all together, which then can be used to determine the average behavior of a single molecule. However, this has changed over the last years. This change comes from new techniques, called single-molecule techniques, that enable researchers to study individual molecules as proteins, lipids, nucleic acids, and other biomolecules in great detail. (Zhao et al., 2013) Those single-molecules techniques could give us answers to those long standing scientific questions.
This research is a first step into answering some of those questions. The aim of this research is to measure the exonulease velocity of T7 DNA polymerase (DNAp) activity, the enzyme and protein that catalyzes the DNA replication process, with the single-molecule technique, Acoustic Force Spectroscopy. It is already shown that this can be done with optical tweezers. (Hoekstra et al., 2014) However, this research aims to find out whether it is possible to use AFS to research processes like this. Getting a deeper, more detailed, understanding of this important biological process is important for scientific and medical advancements.
The structure of the thesis is as follows: first the motivation and relevance of this research project will be discussed. The number one reason most of our loved ones will die is because of chronic diseases like cancer and cardiovascular disease. (Bray et al. 2018; Roth et al. 2017) Research like The Global Burden of Disease Study 2010, the largest systematic review in the world to quantify the world’s health risk factors, worked on by almost 500 scientists, clearly shows that most deaths and diseases in the world are related to lifestyle factors..(Lim et al., 2010) Since I feel many people are unaware of those studies, unaware that they have their health largely in their own control, I wanted to shed bright light on this important case. After this, a brief introduction to the single-molecule
technique AFS as well as a brief overview of the researched protein, T7 DNA polymerase, is
discussed. Next, the experimental set-up will be discussed as along with the methods of preparing the flow cell for the measurement and how the velocity of the DNAp is measured. After that, the results of the measurements and analysis are shown. This thesis is ending in a conclusion and discussion of those results.
1.1 Motivation and Relevance
Why do we even want answers to questions as: “How is DNA replicated”? Not just for the sake of doing science, but because it also has the potential to save millions of lives. All life depends on their genetic code, and how this is translated, stored, retrieved, and replicated. (Alberts et al., 2015) In 2015, the Nobel Prize for Chemistry was awarded to Tomas Lindahl, Paul Modrich, and Aziz Sancar, “for having mapped and explained how the cells repair its DNA and safeguards the genetic
information DNA repair”. (Nobel Prize, 2015) During the award announcement, Tomas Lindahl said the following “To provide better treatment and better [cancer] drugs, we of course have to understand how DNA is damaged.” (Naik et al., 2015)
In 2016, the World Health Organization showed that before the age of 70 years old, cancer is the leading or second leading cause of death in 91 of 172 countries, with third and fourth leading cause in 22 other countries. As can be seen in figure 1 from the world Health Organization, cancer is the leading cause of death for most western and first world countries. Europe accounts for almost a quarter (23.4%) of all cancer cases worldwide, while its population is only 9%, which means that a large percentage of Europeans is confronted with cancer in their lifetime. (Bray et al., 2018)
Figure 1: Data from the World Health Organization (2016) about the incidence of cancer worldwide. This figure shows the ranking of cancer premature mortality per country. In the blue filled countries, cancer is first leading cause of mortality. Light blue, second leading cause. Orange is third or fourth cause. Red is fifth to tenth cause of mortality. (Bray et al., 2018)
As Lindahl said, understanding DNA damage and the DNA repair mechanism can help develop more advanced cancer treatments. DNA repair is a biological defense mechanism with the purpose of protecting the integrity of the genome, which includes genetic material, the genes and chromosomes. (Berwick et al., 2000) DNA repair occurs in human cells, it is a response to DNA damage. DNA damage can happen because of external factors, for example when environmental
chemical substances are inside an organism that are not naturally present, like pesticides, food
additives, carcinogens (cancer promoting compounds), drugs, or environmental pollutants. In addition, internal factors contribute. For example, damage to our DNA can happen from natural biological processes, like oxidative stress (a by-product from ATP creation) or sudden disintegration of chemical bonds in our DNA. (Spits et al., 2003)
Why it is so important to understand this process in detail is because it is found that our DNA repair capacity is largely determined by environmental and lifestyle factors. Research that looked at mutagen sensitivity, which is an indirect way of measuring DNA repair capacity, compared the mutagen sensitivity of twins to compare the heritability of DNA repair capacity. It was found that only roughly 40% to 60% of mutagen sensitivity is genetically heritable, which could mean that half of our DNA repair is influenced by factors that we have indirect and direct control over, as environmental and lifestyle factors. (Wu et al., 2006)
What food we eat might be one of those lifestyle factors that have an impact on our DNA repair capacity. DNA damage occurs frequently at an estimated rate of roughly 800 DNA damages per cell per hour, which is almost 20.000 times that our cell’s DNA is damaged per day. (Vilenchik et al., 2000) Under normal circumstances, the human body is perfectly capable of repairing this damage. However, there are instances when this process does not work properly. Although, DNA repair is utterly complex, and not yet completely understood, many human epidemiological studies have compared the DNA repair capacity of healthy persons with those that have cancer. Many have found an association between impaired DNA repair capacity and cancer susceptibility. (Berwick et al., 2000)
During DNA replication, errors occur that could lead to DNA damage, and the reaction products that result from enzymatic reactions, for example free radicals, can have detrimental
consequences for many biological processes that could negatively impact our health.Research clearly shows that an accumulative damage in cells and organs, for example DNA damage, is associated with functional decline and ageing. (Freitas et al., 2011).
Data from the International Agency for Research on Cancer (IARC) from 2018 shows the estimation of incidences of cancer of 185 countries for 36 types of cancer for all cancer sites combined, based on the GLOBOCAN 2018 database. This data shows that in 2018, there were 18.1 million new cancer cases, and 9.6 million cancer deaths. (
Bray et al., 2018
) However, those numbers might not even be the biggest concern. What might be an even bigger problem is that the International Agency for Research on Cancer has estimated that the amount of new cancer incidences will almost double in the next 20+ years with a 1.6-1.7-fold growth to approximately 29.5 million cancer incidence, and 16.4 cancer deaths per year in 2040 worldwide. (IARC: Cancer Tomorrow, 2018) Moreover, there is another big reason for a better understanding of DNA and DNA repair as it contributes to the development of new cancer treatments or preventive methods. Although some medicines seem to work relatively well, cancer medication frequently comes with high expenditure costs. The fact that expenditure on cancer medicines outgrows the rates of growth of patient population and overall health expenditure, indicates that total cancer costs might become an even bigger problem in the future. (Pricing of Cancer WHO) This is already noticeable in recent health care numbers. While in 2012 global health care expenditures for cancer was a staggering $90.9 billion US dollar, (Global oncology trends, 2017) this cost increased by 46,3% in just 5 years by 2017 to $133 billion US dollar (Global oncology trends, 2018). As can be seen in figure 2 from IQVIA institutefrom December 2017, it is estimated that by 2022, total cancer cost for oncology therapeutic medicines will be between $180 and $200 billion US dollars if this growth continues, which is expected. This can have catastrophically socio-economic consequences if no solution is found.
Figure 2: From IQVIA: the worldwide growth in total cancer cost for oncology therapeutic medicines is illustrated from 2013 till 2017, with future projections for 2018 till 2022, during which the estimated total oncology expenditure of $180 US dollars increases to $200 billion US dollars. (Global oncology trends, 2018)
With cancer being one of deadliest diseases in the world, and with cancer costing over 100 billion dollar per year, soon over 200 billion, finding new and better treatments to prevent those deaths could potentially save millions of lives every single year. This is why it is crucial for humanity that a deeper understanding for DNA, and DNA processes like DNA repair and DNA replication with proteins like DNAp, is acquired.
This research project is the first step in a much bigger process of finding better cancer cures. With this first step, to find out whether AFS is a viable method to research DNA replication. This is done by determining the activity of DNAp, which then is compared to the already established literature values from optical tweezers. Step 2 is to find out if DNA replication, but also translation and transcription can be researched in more detail. For example, if DNAp can be researched well with AFS, then RNA polymerase, the enzyme that catalyzes DNA transcription, could possibly also be researched with AFS. If this is possible, step 3 would be to research better cancer prohibiting medication that binds to ssDNA and dsDNA. For example, the cancer medication “Cisplatina” is one of the most used cancer drugs. (The American Society of Health-System Pharmacists, 2019) Cisplatina binds to the
DNAstrand in a way that it prevents the DNA to replicate, which prevents the cancer cells to grow. AFS might be a very effective research technique to improve medication. As it could screen potential medication fast and effective.
1.2 Acoustic Force Spectroscopy
Currently, single-molecule techniques are available. To answer the research question, to determine the velocity of DNAp, it was possible to use 2 types of single-molecule techniques, optical tweezers and Acoustic Force Spectroscopy (AFS). With optical tweezers a microsphere (bead) is trapped with a laser. This method allows to establish a force on the molecule tethered between two beads and measure this applied force. With optical tweezers it is a feedback loop that keeps the force constant. This feedback loops means that, for a constant force, the distance between the two ends of the DNA needs to be adjusted. This distance change needs to be in proportion to the progress of the enzyme, in our case, DNAp. Because when the DNA starts lengthening, the two ends of the DNA must be changed accordingly to the change in length of the DNA under tension. (Wuite et al., 2000) DNAp is an enzyme that catalyzes the synthesis of double stranded DNA (dsDNA) from single stranded DNA (ssDNA) template and vice versa. (Bustamante et al., 2011)
For my research, I have used AFS, which is a single-molecule technique that combines traditional force spectroscopy with an acoustic manipulation device. I choose this technique because with AFS it is possible to exert a force clamp on hundreds of molecules that are tethered between a surface and a bead in the range from sub piconewtons to hundreds of piconewtons, which is enough force to seriously stretch DNA, alter molecules, and unfold proteins. (Sitters et al., 2015; Bustamante et al., 2011). The force is generated by putting a voltage difference over the piezo element. This in turn, resonantly excites a planar acoustic standing wave that exerts a force on microspheres in the flow cell. This acoustic force can be calculated by formula 1 (Sitters et al., 2015):
(1)
A microsphere with volume V, is subjected to this standing wave experiencing a force F along the vertical (z) direction. In which, p is acoustic pressure, v is acoustic velocity, with ρ⭒= ρp/ρm, the density
ratio and κ⭒ =κp/κm, the compressibility ratio between the particle and the medium. (Sitters et al., 2015)
This acoustic force is exerted on the beads, that are tethered to DNA, and are flushed in the fluid chamber of the AFS chip (Figure 3A), via a pressure difference produced by the syringe that is attached at the top right of the flow cell (3C). This is done by placing the AFS chip in the Flow cell from Lumics with serie number VUFC A5-3. (Figure 3B. When the flow cell is closed with the AFS chip in between (3C), the AFS chip can receive fluids, via the little fluid holder on the middle left, and can receive electrical signals. The flow cell can be connected to a function generator and computer, this way a voltage difference can be put over the piezo element, that generates the acoustic waves. This in turn puts a force on the microspheres, and so the beads start to move. Consequently, because the DNA is attached to the beads, the DNA will stretch. How this works in detail, including the experimental set-up is discussed in the Methods Section.
Figure 3: This figure illustrates the AFS technique with 3 digital camera pictures from the flow cell that is used in this research project. (A) An image of the AFS chip, this chip contains a fluid chamber (2) between 2 glass plates with on top a piezo element (1). (B) The flow cell, with the AFS chip (3). (C) The flow cell closed with the AFS chip in between. Now, the flow cell can be used to do measurements. With the fluid holder (4) where the fluids are flushed in, because of a pressure difference generated by the syringe. (5)
With this AFS set-up, the DNA can be stretched and altered. DNA deformability, how the shape of the DNA changes under applied forces, is valuable to understand because DNA is deformed in many different processes in our cells, as DNA replication, DNA repair, DNA transcription, and many more biological processes that are critical to unravel if one wants to acquire a deep understanding in biosciences. (Bosaeus et al., 2012) In the future, this knowledge could potentially be utilized to improve human health, as is talked about in depth in section 1.1.
1.3 T7 DNA-Polymerase
The genetic blueprint that we inherit from our ancestors is made out of genes. Genes are biochemical information, with each set of genes standing for a specific human trait. It can be seen as a blueprint for the human body, so that it knows how to function, grow and develop. (Alberts et al., 2015). The genotype, the specific set of genes and to a lesser extent non-coding DNA, is mostly what makes every human unique and genes account for many of our characteristic traits like hair, eye and skin colour, but it may also play a large role in our behavioral characteristics as intelligence, way of
communicating, and our talents. The total set of genes in an organism or a cell is known as the genome. Three billion base pairs (bps) of DNA is the estimated amount that is packaged into each of our 46 chromosomes, which consists of long double stranded DNA helix that makes up the human genome. (Venter et al., 2001) For human growth and development, cell division is needed, a copy must be made from every gene in the genome. It is highly important that this replication occurs in a correct way, to preserve and pass on the genetic code correctly. The special enzyme, T7 DNAp can do this with remarkable speed and efficiency. DNAp reads one strand of DNA and synthesizes a new strand exactly the same as the original, except possibly for a few errors. This way both daughter cells will get a copy of the original DNA. (Alberts et al., 2015) It has also been shown that T7 DNAp can do this the exact opposite way, meaning that DNAp can make single stranded DNA (ssDNA) from double stranded DNA (dsDNA). This process is called exonuclease, which is the process at which the velocity of DNAp is determined in this research project. (Donlin et al., 1991) This acquired data is
then compared with the literature values acquired by Hoekstra et al. 2014, which is shown in figure 4. This data is acquired with the already established single molecule technique: Optical Tweezers.
Figure 4: Image from Hoekstra et al. (2014) DNAp activity for polymerization (black) and exonuclease (red) at different constant forces. The rates of polymerization seem to decrease with increasing tension while exonuclease activity does not seem to differ a lot with higher tension.
The need for correctly replicating the genetic code is satisfied by DNAp for three important mechanisms. First, its selectivity for the correct nucleotide to be incorporated, DNAp catalyzes base pairs relatively fast, with an error of approximately one in 105-106 base pairs. Secondly, after a miss
duplication happens, DNAp can recognize this mismatch and cut out the error. Thirdly, DNAp can self-correct this mismatch by replacing the error, the cut out base, with the right one. DNAp can repair those mis incorporated bases for all but one out of 103-104. This shows that DNA polymerase is
extremely effective in DNA replication with only one error in approximately 108 to 1010 base pairs.
(Johnson et al., 1993)
2. Methods
2.1 Experimental Setup
The goal for this experiment was to measure the velocity of DNAp. For this experiment an acoustic wave force is used to stretch the single-molecule, DNA. This is done with the AFS setup that can be seen in figure 5. A led light with 455 nanometer wavelength is used to produce an image of the bead
that can be seen in figure 6C. This led light goes through the AFS chip, and the image is created with use of an objective lens, and digital CMOS (complementary metal-oxide semiconductor) camera.
The flow cell contains the DNA and is prepared with a specific protocol that will be discussed in detail in the next section. This flow cell also contains a piezoelectric sensor, that can convert electrical signals to different forces. This piezo element is connected to the computer with a build in function generator, this is connected to a Generation 1 amplifier from Lumics, and a transformer. The function generator is connected via USB and is operated with the program, LabVIEW 2016. A planar standing wave can be generated in the piezo element with the function generator that is altered by the connected amplifier.
The DNA in the fluid chamber of the flow cell can attach to the upper or lower glass plate and a polystyrene microspheres of 4.5μm diameter as is illustrated in figure 6B. Also, smaller beads of 1.9μm diameter are used that don’t have DNA attached to them, but those beads bind with the anti-dig on the surface. This is to acquire an accurate measurement of the drift. This way there can be
differentiated between the beads that have DNA attached to them and those that do not. This will be discussed in more detail in the results section. The planer standing wave exerts a force on the beads, which moves the beads, and stretches the DNA strand.
Figure 5: Modified image from, Kamsma et al. (2016). This figure is an illustration of the AFS setup that is used for this research project. (I) From top to bottom, a LED light with 455 nanometers, goes through the AFS chip to create the image utilizing an objective lens. Then, the LED light beam is received by a digital CMOS camera. (II) The flow cell consists of two glass plates with in between a fluid chamber. On the upper side of the glass plate, a transparent piezoelectric element is attached that is electronically connected. This piezo element can measure force differences and convert this into electrical output. (III) This piezo element is connected to computer via, the function generator together in series with the amplifier, and a transformer. (Kamsma et al., 2016)
Figure 6: Modified image from, Sitters et al. (2015). In this figure, a more microscopic level can be observed on how the experimental setup works. On the left can be seen schematic overview of the flow cell, with a piezo element, and two glass plates with in between a fluid chamber. But the middle image is a zoomed in schematic image of how the DNA is attached to a bead. Here is a single DNA molecule attached to the upper glass plate, illustrated with the black stars, with the upper end of the DNA strand, and the DNA is attached to the bead with the lower end of the DNA strand. The DNA strand can now be stretched by exerting acoustic forces on the bead. In the right image, a digital picture can be seen, with a view from the x-y axis of the flow cell. The upper left bead is a large, 4.5μm diameter bead and on the lower right a smaller 1.5-um diameter bead can be seen. (1.9μm is used in this research) (Sitters et al. 2015)
2.2 Flow Cell Preparation Method
In the flow cell, the fluids get flushed in the fluid chamber. Here, an electric voltage can be put over the piezo element, which stretches the single DNA molecules. First, the flow cell need to be cleaned from any substances that were flushed in before. Start with flushing with NAOH (Sodium Hydroxy) or KOH (Potassium Hydroxide), let it incubate overnight to remove sigmacote from previous
measurements. Those two substances are strong bases that can release OH-ions and can promote certain chemical reactions that have a base as catalyzer, like DNA replication. Next, flush with 200μL ultrapure water, MMQ (milli-Q). Then, incubate bleach for at least 20 min to remove all beads and substances from previous measurements. After this flush with MMQ again. Next, incubate and refill the flow cell with 0.5Mol Na2O3S2 (Sodium Thiosulfate) for 25 minutes to make sure all bleach is
removed. Then, flush with MMQ again and finally flush without any substances, but only with air. Now the surface must be made hydrophobic. This is done by flushing in 100 μL Sigmacote, incubate for 1 minute, and follow it with flushing air. Next flush MMQ, also followed by flushing air. Then, flush with acethon, and flush with air again.
The surface must now be made functional, so that a measurement can be done. To
functionalize the surface, first flush with (phosphate-buffered saline) PBS. PBS helps to keep the pH grade constant and helps to replicate the internal conditions of the human body, as ion concentration for example. Next Incubate Anti-DIG with 20μg/mL in PBS for a minimum of 25 minutes. Then, incubate and refill the flow cell with 0.5% pluronics + 0.5% BSA for at least 30 minutes. After this
flush with Blothing Grade Blocker (BGB) buffer. This BGB buffer consists of 10 mMol Tris-HCl pH7.5, 150 mMol NaCl, 1 mMol EDTA pH 8.0, 1 mMol DTT, and 3% Glycerol. Now, incubate with 4% (weight/volume) BGB in BGB buffer for 10 min minimum. Before the beads are flushed it is important to flush first with a specific measurement buffer. The measurement buffer used for this research is a combination of 10 mMol Tris pH7.5, with 50 mMol NaCl, and 8 mM MgCl2.. Next,
incubate the 1.9μm DIG-beads for 10 to 15 minutes, check if the beads are properly stuck to the surface with LabVIEW 2016 and flush the loose beads away with light pressure. If there are not a minimum of 30 to 40 1.9μm beads stuck, incubate for 15 minutes longer or clean with bleach first, and redo the functionalization, depending on how optimal the surface is. If there are 30 to 40 1.9μm beads stuck, flush with MB until almost all loose beads are gone. Then, incubate 70μL of PKYBI DNA with a ratio of 1:4000, for at least 30 minutes. Next, incubate 1μL of 4.5μm beads coated with streptavadin for a minimum of 20 minutes. Now, flush the loose beads away and check if at least 50 to 60 4.5μm beads are properly stuck. If not, incubate 10 minutes longer or clean with bleach, and redo the
functionalization process of the surface. If there are at least 50 to 60 4.5μm beads stuck, flush with MB until almost all loose beads are gone. Finally, 0.8μL of DNAp in 50μL of MB can be flushed in. Now, the DNAp measurement can begin.
2.3 Method of Bead and DNAp Measurement
If one wants to know the velocity at which DNAp makes ssDNA from dsDNA, or vice versa, it is important that the displacement of the 1.9μm beads, and 4.5μm beads that are attached to DNA, are accurately measured. In this research project is LabVIEW 2016 used to measure bead and DNA displacement. The measurement in LabVIEW goes as follows. First, the resonance frequency must be found because this is the frequency at which the flow cell responds ideally to the electrical voltage. This is determined by finding the maximum output signal, when a frequency sweep is set over the piezo element. Then, assign the beads manually, because this is often more accurate than if the beads are assigned automatically by LabVIEW. Assign 40 1.9μm beads first, after this assign 60 4.5μm beads. This ratio of a 40/60 seems to be the ratio for accurate measurements. In figure 7, an ideal field of view can be seen that is acquired in this research project on 8 May 2019. To make sure LabVIEW tracks the beads precisely, it is important to create a Look Up Table (LUT) with fixed steps of 100nm from 0 to 8000 nanometer. Next, preview the settings, and make sure that height determination graph is at ~7000 nanometer. This represents the starting point of the measurement. Now, the beads can be recorded accurately.
Figure 7: An ideal field of view from above the flow cell, made with the AFS experimental set-up used in this research project as talked about in the previous section. Image made on 8 May 2019. An ideal ratio of 40 smaller 1.9 μm beads and 60 larger 4.5 μm beads can be seen.
2.4 Method of Calibration: Conversion Factor from Voltage to
Force
With the used AFS set-up it was not possible to directly put a known force on the beads. This force needed to be calculated. However, with this set-up a known voltage could be put over the piezo element, and via this piezo element exert an acoustic force on the beads, as discussed in section 1.3. But this force is difficult to calculate. This calibration is done to find the acoustic force that is placed on the beads in another way than with formula 1. The calibration method used is called, shooting beads calibration, and for this we need to know the other relevant forces that exert some force on loose beads. A schematic overview of these forces can be seen in figure 8, a loose bead without DNA, not tethered to the surface, can be seen. In the beginning, there is no voltage set over the flow cell, the bead is on the surface of the lower glass plate (Capping), then a voltage is put over the flow cell, which generates the acoustic waves, and puts a force over the beads, so the bead moves upward. Then 1-2 seconds later the function generator is turned off, so the bead does not experience the acoustic force any longer. As can be seen in figure 8, the beads slowly start to fall down to the lower surface again.
Figure 8: (Figure adjusted from Kamsma et al. 2016) The vertical-axis is the z-displacement. The
horizontal-axis represents the time. The forces that act on the free microspheres in the fluid chamber are the force of gravity (FGrav), force of buoyancy (Fbuoy), the
Stokes drag force (FDrag), and the acoustic force (=
Acoustic Radiation Force: FRad in figure 8)
The forces that act on the bead in this calibration process here are not just the acoustic force Also, the force of gravity. The force of buoyancy, because of density differences. And the Stokes drag force. Figure 9 illustrates the path of the bead because of those forces together in practice. In figure 9, a screenshot
from data acquired with LabVIEW in this research project at 24 May 2019 can be seen. As can be seen from the data in figure 9, when the voltage (pink line) is turned off, the bead’s position falls back with roughly a straight line till its baseline level, back to where it started. This straight line means the rate of change in distance is constant, which means the velocity is constant.
Figure 9: This is a screenshot from the program LabVIEW that is used in the research project. This data is acquired at 24 May 2019. Left vertical-axis, the distance, the green line corresponds with the vertical (z) distance of the bead. The horizontal-axis represents the time in minutes. Lastly, the pink line corresponds to the voltage, which is divided here by 10, the right vertical-axis. This figure illustrates nicely the path of a loose bead that is subjected to the acoustic force, force of gravity, force of buoyancy, and the Stokes drag force.
If the velocity is constant, this means that the acceleration is zero, and the total force acting on the bead is zero as well. This knowledge gives rise to the following formula (Kamsma et al., 2016):
With,
(3)
(4)
(5)
For the gravitational force, V is the volume of the bead, ρp is density of particle, which is the density of
the bead here, and g is the gravitational acceleration. Those are all known constants, so the force of gravity is constant. For the buoyancy force, V is volume of bead, with ρm the density of the medium,
which is roughly the density of water. For the Stokes drag force, ɣfaxen is the effective drag coefficient,
also a known constant. (Schäffer et al., 2007) So the only thing left needed is vp , the velocity of the
particle upwards when the voltage is put over the bead, here the upward velocity of the bead. This upward velocity is calculated with analyzing the part when the voltage is put over the piezo element. Because with LabVIEW the vertical (z) distance is measured, together with the time at a corresponding beforehand known voltage, the velocity can be calculated at each voltage. To analyze this data, I have used LabVIEW and a python script. The result of this calibration can be seen in figure 10, where the Acoustic Force is on the vertical-axis and the voltage squared on the horizontal-axis. This relationship should be linear because the acoustic pressure is linear with the voltage. And the acoustic force is related to acoustic pressure squared. So, the force must then, also be linear with voltage squared, this line is plotted through the points in figure 10.
Figure 10: The linear relation of acoustic force with voltage squared. With acoustic force on the vertical-axis and voltage squared on the horizontal-axis.
3. Results and Discussion Analysis
The end-to-end lengths of dsDNA and ssDNA are the same length when very small forces are applied to the DNA strands. However, the strands are significantly different at tensions of 6pN or higher. As can be seen in figure 11, with exonucleolysis (nuclease activity; the dashed red line), dsDNA (black line) is made into ssDNA (red line) by DNAp, the extension moves from the black line, to the red line, and the DNA strand is elongated. This process also happens opposite direction if the DNAp goes from ssDNA to dsDNA, called polymerization.
Figure 11: Image from Hoekstra et al. (2014). Force-extension curves are illustrated for dsDNA (black) and ssDNA (red). At the same force, it can be seen that the length of base pairs are significantly different for dsDNA and ssDNA above 6 piconewton (pN) The black dashed arrow represents polymerization activity by DNAp, the synthesis of dsDNA from ssDNA, which leads to shortening at constant tension. The red dashed arrow represents exonucleolysis (nuclease activity by DNAp), from dsDNA to ssDNA, which leads to lengthening of the DNA strand.
The DNA lengthening is measured with LabVIEW 2016. The raw x,y,z traces are drift corrected to exclude apparent lengthening due to external factors, such as drift of the stage. To do this, displacement of the 1.9μm beads must be subtracted from the displacement from the DNA tethered 4.5 μm beads, to get a correct measurement of how much the displacement of the DNA was, and so how much the DNAp activity was. In LabVIEW, the x, y, and z position of the beads tethered with DNA are tracked over time at constant forces between 3 pN and 25 pN. With knowledge about the position of the beads and the time period in which this displacement happened; the velocity can be determined. In figure 12, data from 8 May 2019 is shown; here exonuclease activity can be seen at a force of 20 pN. Moreover, because the displacement is not a single straight line over time, the curve is divided in different areas, represented by different colors, to acquire an accurate representation of the
different velocities of DNAp. The velocity rates, in which DNAp makes ssDNA from dsDNA, are given in micrometer per minute at the different areas, which are shown next to the curves.
In figure 11, at around 20pN, beginning with dsDNA is an extension of 0.325 nm/bp. During exonuclease, DNAp turns this into ssDNA, which at 20pN is an extension of 0.475 nm/nt, which gives an extension of 0.150 nm/bp. This extensions allows to give the velocities in base pair per second to verify this AFS data with existing OT data from Hoekstra et al. (2014). This is the activity, measured in velocity, of DNAp, because when DNAp makes ssDNA from dsDNA, the base pairs are extended. In figure 12, this results in 4.9bp/s, 13.4 bp/s, and 3.9 bp/s, for the red, blue, and green points, respectively.
Figure 12: A z-position vs. time curve for a single bead p at 20 pN , acquired on 8 May 2019. The colored areas each represent a linear slope, i.e. a constant velocity, divided by eye. The velocities are shown in their matching color next to each curve in μm/s.
To answer the research question whether AFS is a viable method to research DNA replication, more specifically, DNAp exonuclease activity, the data needs to be validated by previous research. As mentioned before, it is established that the exonuclease velocity of DNAp can be accurately measured with optical tweezer, as demonstrated by Hoekstra et al. (2014). In figure 13, a histogram of the exonuclease (red bars) velocity, as well as the polymerization velocity (black bars) of DNAp at a constant force clamp of 20 pN is shown. From this, it is clear that the majority of the exonuclease velocity of DNA is between 0 and 250 base pairs per second. So, the found velocities in figure 12 correspond nicely with this distribution found by Hoekstra et al. (2014).
Figure 13: Image from Hoekstra et al. (2014). Histogram from DNAp velocities measured with optical tweezers. Both exonuclease activity (red), and polymerization activity (black) are shown here, with on the vertical-axis the amount of data points, and on the horizontal-axis the DNAp velocity in rate of nucleotide per second.
Although the data found seems to nicely fit the expected values from literature, this is only a preliminary conclusion, as the number of data points is low. More data needs to be acquired to make a definite conclusion on whether AFS is a viable method to research processes like DNA replication, and determine DNAp activity. However, a lot more measurements were done, but many did not lead to high quality data. This low yield of clear DNAp activity data could be due to various reasons.
First of all, certain circumstances prevent you from starting a measurement at all. Sometimes, air bubbles come into the flow channel, and when those air bubbles are flushed in while the beads and DNA are stuck to the surface, the air bubble will flush the beads away. Then, the flow cell needs to be prepared again, which reduces the number of measurements per day. This can be minimized by flushing slowly and making sure the fluid holder is tightly placed in the flow cell.
Secondly, it can occur that the 1.9 μm bead and DNA do not bind well to the surface. In figure 14, a schematic illustration is shown, of how beads bind to the DNA and the surface. In this figure can be seen that the 1.9 μm bead has a dig coating (green) over the surface, this binds well to the anti-dig (pink) that is supposed to bind with the surface. If the bead does not bind well to the surface, it is probably because of a problem with the anti-dig coating: if the anti-dig stock is faulty, a fresh anti-dig stock solution should reduce this. Also, the beads and DNA can be incubated longer to counter this problem as much as possible.
If those problems do not occur, the measurement can be started. However, problems can also happen with the measurement. For example, beads and DNA can bind non-specifically, meaning that they do not bind with each other, but with other things in the fluid chamber. For example, the DNAp
can bind to glass. This would reduce the effective concentration of DNAp and lower the number of DNA molecules that have DNAp bound and thus reduce the number of data points per measurement. The binding of DNAp to glass should be prevented by the passivation of BSA and pluronics. As can be seen in figure 14, BSA and pluronics bind directly to the surface because those substances bind stronger to the surface than the beads or DNA for example, this way they block other molecules that want to bind to the surface. Moreover, if not enough BSA and pluronics bind to the surface, lots of aspecific binding can occur as that DNAp, DNA or the beads bind directly to the surface, which is hindering the measurement.
However, this passivation has been ineffective during some measurements, as some had a lot of aspecifically bound beads to the surface. This ineffectiveness could be caused by an underlying surface issue, and in that case, it makes sense that the anti-dig coating is also not optimal.
Figure 14: Schematic image of beads tethered to the lower glass surface in the fluid chamber of the flow cell. The 1.9μm bead has a dig coating that binds with the anti-dig, which in turn binds to the surface. The 4.5μm bead has a streptavidin coating that binds to the biotin coating on the end of the DNA strand. On the other end of the DNA strand, dig is binding with the anti-dig on the surface. Two DNA strands are attached to the 4.5μm bead, which indicated that the DNA concentration was too high.
Moreover, it can also be the case that multiple DNA strands bind to the same bead (figure 14), when the DNA concentration is too high. Then, DNAp activity on one of the strands will not result in clear nor reliable data. A too high concentration could have been used because it first seemed that it was the right concentration to get a good amount of tethered beads. It is possible that the streptavadin coating on the beads (yellow) is not uniform anymore. This coating normally binds to biotin (blue), which is on one end of the DNA strand. This hypothesis can be investigated by taking another bead stock and see what the optimum DNA concentration for these beads is.
In addition, it could be that the DNAp does bind to the DNA but is not active. However, this is not very likely as the DNAp is recently purchased, and because of that not degraded.
Finally, it could be the case that the force is not constant, because if the wrong frequency resonance, which is temperature dependent and most determining for the force, is used then the force will not be constant. A standing wave cannot be generated with the wrong resonance frequency. However, the temperature difference during the measurement has been tracked, and the temperature was constant throughout the measurements, so the resonance frequency would be constant over the measurement.
4. Conclusion
The revolution in science with the coming of single-molecule techniques, so that molecules can be tracked and measured accurately individually, brings many new opportunities. However, single-molecule techniques like optical tweezers can merely measure one single-molecule at the same time, which makes the research progress slow. The faster and accurate research can be done, the faster science can develop, and new medication can be found. That is why in this thesis, the aim was to find whether this single molecule research can be done one a multiplexed scale, for increased scientific and medical advancement. Medical advancement is highly needed because in most western countries, the number one leading cause of death is cancer, with almost 20 million new cancer cases every year, which is expected to double in the next 20+ years. Moreover, cancer costs are projected to grow to roughly 200 billion US dollars in only the next couple of years.
For those reasons it is important to find out more about DNA and the DNA replication process because cancer is correlated to DNA damage, which can come from errors in DNA replication.
Furthermore, cancer medication that binds with ssDNA and dsDNA exists, for further development it is crucial to get a deeper understanding of ssDNA and dsDNA processes that this medication
influences. For this research project, the exonuclease velocity of DNAp, which catalyzes DNA replication, is determined with AFS, to find out whether AFS is capable of researching DNAp. As a preliminary conclusion, it seems that accurate DNAp activity can be measured, because the acquired data is in accordance with the known measurements from optical tweezers. However, for a definite conclusion, more data at different forces is needed. To acquire more data with future experiments, several elements need improvement. Firstly, the right concentration of DNA must be established, by testing different concentrations and stock samples. Secondly, the surface must be made optimal by making sure that the anti-dig and coating is present. When AFS is indeed capable of measuring DNAp nuclease activity accurately, future research would be to observe polymerization and to include the addition of ssDNA or dsDNA binding proteins or medicines to observe their effect.
