Determining the Interaction of Cresyl Violet Acetate using Computer Modeling and Spectroscopy

Table of Contents

Abstract

Chapter 1 : Introduction
1.1 Spectroscopy
1.2 Ultraviolet Visible Spectroscopy
1.3 Fluorescence Spectroscopy
1.4 Nucleic Acids
1.5 Modes of Interactions: Intercalation
1.6 Cresyl Violet
1.7 Scatchard Plot Analysis
1.8 Computer Modeling: Molecular Operating Environment

Chapter 2: Experimental
2.1 Computer Modeling
2.2 Fluorescence Analysis
2.3 UV-Visible Spectroscopy Analysis
2.4 pH Analysis
2.5 Scatchard Plot Analysis

Chapter 3: Results/Discussion
3.1 Computer Modeling
3.2 pH Experiments
3.3 Fluorescence Effects of Cresyl Violet Bound to DNA
3.4 Scatchard Plot

References

Abstract

Science is always finding new innovative ways to help cure diseases like HIV, cancer, and Alzheimer’s. Fluorescent dyes have been a way to trace certain genes within our DNA (deoxyribonucleic acid). It has been brought to our attention that cresyl violet acetate, an organic compound, stains blue when working with DNA, but stains purple when working with RNA. Cresyl violet also changes color based on differences in base pairing.

With the increasing use of technology, computer modeling and spectroscopy have been used to analyze the interaction of cresyl violet when bound to DNA. The Molecular Operating Environment (MOE) computer modeling software was used to predict possible modes of interaction. The results were fairly constant, showing backbone interactions with the amine groups of cresyl violet. There was also hydrogen bonding predictions between cresyl violet and DNA. Intercalation, or π-π stacking was only predicted for adenine ssDNA with cresyl violet. Cresyl violets cationic character, allowed for pH to play a factor in its binding ability, and therefore it was determined that pH held at 7.2 - 7.4 were the best conditions. Dimerization was controlled using both low concentrations of cresyl violet and γ-cyclodextrin. By tracing pH and dimerization we can compare results to docking. This is since docking was set for cresyl violet with a +1 charge and with monomers docking to the DNA.

Scatchard plot analysis showed that binding constants are affected by concentration of cresyl violet, and the inhibition of electrostatic interactions and dimerization. The lower the concentration, the less binding because of free versus bound ligand availability. At high concentrations, dimerization is too high, inhibiting binding. The use of spermidine to prevent electrostatic interactions, proved to improve binding of cresyl violet to DNA. γ-Cyclodextrin sequesters dimerization, which also improved number of binding site. This could suggest intercalation as a possible mode of interaction because more binding is present when both interactions are unavailable, however, more experimentation is needed to conclude that intercalation is in fact present.

Chapter 1: Introduction

Understanding the interaction of small molecules when bound to DNA has been an interest in scientific research due to its importance in DNA tracing and new more efficient drug discoveries [1]. There are several binding modes that can occur including electrostatic interactions, groove binding, and intercalative binding [1]. These binding modes can each be better investigated using different types of spectroscopy. Atomic and molecular structures are well known by studying how they interact with light [2].

1.1 Spectroscopy

Spectroscopy is the study of the interaction of matter and light, and occasionally studying the interaction of matter and other forms of energy [6]. Depending on where the energy lies on the electromagnetic spectrum, it can decipher what kind of information can be told by the interactions [2]. Spectrometry is used and referred to as measurements of the intensity of radiation with a photoelectric transducer or electronic device [6]. Radiation is energy in the form of light. To better understand spectroscopy, it is important to understand the properties of light. Light is both wave-like and particle-like. The particle form of light energy is called a photon, a bundle of electromagnetic energy. Photons are proportional to the frequency of the radiation [6]. Electromagnetic energy is described according to the classical sinusoidal wave model, which describes wavelength, frequency, velocity, and amplitude. Frequency is inversely proportional to wavelength.

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Where c is the speed of light, λ is wavelength in nanometers, and v is frequency in Hertz. In the second equation ΔE is the change in energy and h is plank’s constant 6.63 x 10"34 J*S
(discovered through the photoelectric effect). Understanding light as a form of radiation is important when studying spectroscopy. There are several topics on how radiation works; there is diffraction, transmission, refraction, reflection, scattering, and polarization. Diffraction of lights is the process where a parallel beam of radiation is bent as it passes a narrow opening [6]. Diffraction is a wave property and can be easily seen in water, and how waves change when passing through slits. Wide openings have slight changes in the waves, however small openings of the same order of magnitude of the wavelength cause high diffraction, where there is a bend in the wave. Transmission of radiation involves refractive indices. The refractive index depends on the medium and its interaction with light.

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Where n is the refractive index, v is the velocity, and c is the velocity in a vacuum. Refractive indices are lower for liquid and high for solids [6]. It is important to realize that the refractive index changes at varying wavelengths. This is called dispersion. There are normal dispersion regions where the refractive index changes only slightly. Contrary to this, anomalous dispersion regions are where the refractive index has sharp changes. Refraction of light is where there is radiation that passes at an angle through two different mediums with different densities. This is due to the changes in velocity through the different mediums. This is defined by Snell’s law:

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There is also reflection of light. This causes a loss of light that passes through two different mediums with two densities.

Lastly there is scattering of radiation. Transmission of radiation in matter is the retention of energy by atoms, ions, or molecules, which reemit the light in all directions as the particles return to their original state. When particles are very small relative to the wavelength of the light energy, destructive interference removes most of the reemitted energy, however, the energy that travels to the direction of the beam make it appear that there is no change as a consequence of the interaction [6]. The intensity of the scattered radiation increases depending on particle size. There is Rayleigh scattering and Raman scattering. Rayleigh scatter is when the scattered molecules are smaller than the wavelength of the radiation. The intensity due to Rayleigh scattering is proportional to the inverse wavelength to the fourth power. A good example of Rayleigh scatter is why the sky is blue, resulting in the greater scattering of the shorter wavelengths of the visible spectrum [6]. Raman scatter involves polarization resulting in changes in transitions of vibrational energy. There is also polarization of radiation. Polarization is an understanding of light as a wave particle. A photon is shown as vibrating in an oscillating motion starting from zero and going above and below in the height of its amplitude. This can occur, however, in several different planes. Removal of one of the planes of vibration can produce that is plane polarized. This explains radio waves and microwaves.

Since spectroscopy involves light and matter, energy states of different chemicals should be investigated. Max Planck, a German physicist, advised the quantum theory that later helped rationalize more emission and absorption. This shows that atoms, ions or molecules that absorb or emit energy have an energy difference related to frequency or wavelength of energy (equation 2). Energy in the form of light, electrical energy or heat, acts as a stimulus to have the sample become exciting, and the electromagnetic radiation emitted is measured and the sample goes back down to ground state. There are different kinds of spectroscopy. Emission spectroscopy involved when the energy used in heat or electrical energy. Chemiluminescence is when energy is in the form of a chemical reaction. These two spectroscopies can tell concentration of a species. There are other kinds of spectroscopies that are widely known; absorption spectroscopy, ultraviolet-visible spectroscopy, fluorescence spectroscopy, mass spectroscopy, infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance spectroscopy. Each of these spectroscopies can tell us something different about the molecule. These different spectroscopies all are radiated with energy at different ranges. These ranges are depicted on the electromagnetic spectrum (Figure 1.1).

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Figure 1.1 Regions of the Electromagnetic Spectrum [8]

Absorption spectroscopy compares the amount of light before and after it interacts with a sample. Absorption occurs when there is an energy difference equal to the energy of the photons between the different states of the material [3]. Infrared spectroscopy uses light in the infrared region to determine functional groups present [4]. Near infrared spectroscopy is based off of overtones and harmonic vibrations, mid infrared is based off of vibrations that refer to rotational- vibrational structure, and far infrared is low energy for rotational spectroscopy [4]. Raman Spectroscopy is based off of inelastic scattering of monochromatic light. The photons from the light are absorbed by the sample and then reemitted; there is then a shift in the monochromatic frequency. This shift can provide information about vibrational and rotational transitions within the molecule [5]. Nuclear Magnetic Resonance Spectroscopy is used in determining the structure of organic compounds by understanding the carbon and hydrogen framework. Mass spectroscopy is used to determine molecular size and formula. Ultraviolet Visible Spectroscopy and Fluorescence spectroscopy are widely used when working with deoxyribonucleic acids and fluorescent compounds and therefore they will be discussed in more detail.

1.2 Ultraviolet Visible Spectroscopy

Ultraviolet Visible Spectroscopy is a form of absorption spectroscopy. Absorption spectroscopy in the ultraviolet and visible region is widely used for quantitative information of many inorganic, organic, and biological molecules [6]. The ultraviolet region of the electromagnetic spectrum extends from the short-wavelength end of the visible region to the long wavelength end of the X-ray region (4 x 10"7 m to 10"8 m). When molecules are irradiated with photons, the radiation either passes through the sample or is absorbed. Absorption is when electromagnetic radiation is transferred to atoms, ions, or molecules and it promotes particles from their normal state to excited state one or two levels higher [6]. The energy absorbed by the molecule is in relation to the amount of energy necessary to promote and electron from a lower- energy orbital to a higher-energy one in the conjugated molecule [7]. When there is excitation due to energy absorbed, the molecule has a π electron that promotes from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). There can be sigma bond and pi bond transitions. Sigma bond transitions usually require less energy than pi bonds. These transitions are noted as σ -> σ* or π -> π* excitation. The energy between HOMO and LUMO is the energy associated with Xmax. Based off the quantum theory, atoms, ions, and molecules have limited number of energy levels. The energy of the photon must be the exact energy difference between HOMO and LUMO. Every species has a different energy difference. Energy levels are demonstrated in energy level diagrams, the most popular is the Jablanski Diagram. The Jablanski diagram shows absorption energy levels, fluorescence energy levels, and phosphorescence levels, which will be discussed later (figure 1.2).

It is also important to know about Werner

Heisenberg’s uncertainty principle, because this puts limitations in locating the electron and its momentum. Therefore we can determine its location but then cannot be sure of its momentum, and vice versa [10]. Absorption spectroscopy is based on the measurement of transmittance, which is interchangeable with absorbance. The absorbance can be defined as,

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Figure 2. Jablonski Energy Diagram [11]

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Where Io is the intensity of the incident light and I is the intensity of the light transmitted through the sample. Quantitative absorption methods require two power measurements. These measurements are the power to pass through the cuvette to pass through the analyte and the other is to through the other side of the cuvette. The amount of ultraviolet light absorbed can be expressed at the molar absorptivity, ε, and can be defined as,

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Where A is absorbance, c is the concentration in molarity, and l is the path length of the sample in cm. Therefore the units of molar absorptivity is M-1 cm-1. Molar absorptivity is different for each molecule and therefore a physical constant. The equation relating molar absorptivity and absorbance can be written as,

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This is Beer’s law, when a monochromatic radiation is used. Beer’s law is used to determine concentration when using absorbance spectroscopy. However, there are limitations to Beer’s Law. The path length is an importance consideration, because absorbance indirectly realized to path length, but then deviates from frequency [6]. Other limitations are analyte concentrations. When there are high concentrated solutions (> 0.01M) then there is a high amount of molecules bumping into each other causing an influx of solute-solute interactions, solute-solvent interactions, and hydrogen bonding effects. This then allows a smaller amount of light to pass through the analyte, and therefore less energy. This causes error because the quantum theory says the energy has to exactly enough to move the electrons from a ground state to its unique excited state [6].

The measurements done on Ultraviolet Visible Spectroscopy are possible because of the instrument. Spectrophotometers are the instruments that capture the energy associated with light at specific wavelengths. The ultraviolet visible spectrophotometer measures electromagnetic radiation of a solution within the range roughly 200 - 900 nanometers, depending on the instrument. Ultraviolet visible spectrophotometers use the visible region of light spectrum to see what is occurring in the solution. Visible color is the color which our eyes see because the substance can absorb all light but reflects the color light we see. The color of the light reflected is what we see as the color. The actual ultraviolet visible spectrophotometer uses a cuvette to hold the solution. Cuvettes are typically glass, quartz, or sometimes plastic. The cuvette must be able to allow light to pass through it with as little reflection loss as possible. The simplest spectrophotometer has at least five components, including the cuvette. There needs to be a stable source of radiant energy (light source), the device must have a way of restriction regions of the electromagnetic spectrum for measurement, a detector that converts light energy into an electrical signal, and a signal processor and readout that displays the information on a digital screen (computer) [6]. Absorption spectrophotometers have a beam from the source that passes into the wavelength selector and then through the sample (figure 1.3 a.) [6].

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Figure 3 (a) Absorption Spectrophotometer (b) Fluorescence/ Phosphorescence Spectrophotometer (c) Emission Spectrophotometer [12]

1.3 Fluorescence Spectroscopy

Fluorescence Spectroscopy is a form of spectroscopy that is being more and more widely used in recent years. Fluorescence is a subcategory of luminescence. Luminescence is not light energy that gives off heat; it is referred to as “cold light” that is dispersed at lower temperatures. The cold light is due to chemical reactions rather than a heat source, and it is one of the reasons why fluorescent light bulbs are more efficient than using an incandescent light more efficient than using an incandescent light source, which excites atoms through heat. Luminescence is when we have a luminescent substance that is excited by an energy source and has its electron (negatively charged particle) pushed out of its lower energy state into a higher energy state. With few exceptions, the excitation energy is always greater than the energy (wavelength, color) of the emitted light. This is because not all absorbed light is released. The fluorescent process is the idea of being excited by high energy and then giving off energy in the form of visible light. This process of exciting a fluorophore can actually be done by a fluorescent spectrophotometer. Fluorescence and phosphorescence spectrophotometers are similar to absorption spectrophotometers, however, the source induces the sample in the cuvette to emit characteristic radiation that are measured at a 90 degree angle with respect to the source [6]. A fluorescence spectrophotometer uses a light source to excite a sample in a sample cuvette (Figure 1.3), and there are two monochromators, which excite the molecules and record the emission. Monochromators are optical devices that have the ability to narrow or widen to achieve certain wavelengths. When a fluorophore is excited either by using a spectrophotometer or by means of a chemical reaction, there are three stages: excitation, excited-state lifetime, and fluorescence emissions.

During the excitation stage a lamp or laser passes a photon of energy to the fluorophore. The most popular fluorophore is actually quinine, and it is used as the standard for many fluorescence spectrophotometers. A standard is the controlled group when running samples through the spectrophotometer. We do not know the true value of fluorescence intensities of a sample, but we have values relative to a standard. After much study, scientists have been able to detect the shapes of the molecules that typically fluoresce. Fluorophores are predominantly aromatic; meaning that they are organic compounds, which display more stability than a hydrocarbon chains because they are cyclic. An example of an aromatic compound is quinine. The fluorophore then rises from the ground energy state to an excited state. The excited-state lifetime is typically 1- 10 nanoseconds. During this time there are several chemical processes that can happen. The energy stored in the excited state could be released and the molecule goes back to ground state where the fluorescence emission originated, where no fluorescent light is given off and therefore it is not considered a fluorophore. An example of this could be of pure water. When we excite water using a xenon lamp (a source of UV light), the energy absorbed is equal to the energy released and no fluorescent light is given off. Another possibility is when not all the energy is dispersed and the fluorescence emission is not the same as the fluorescence absorption. This is when we see fluorescent colors appear [13]. Quenching is another process that can occur where deactivators, typically ions such as chloride, iodide, and oxygen, do not allow the fluorophore to fluoresce as intensely because they absorb the energy it gives off [13].

The final stage of fluorescence is called fluorescence emission. It was determined by Sir G. G. Stokes in 1862 at the University of Cambridge, where he used a blue window that transmitted light at 400 nanometers. It was absorbed by quinine and then released at 450 nanometers, which is in the range of visible light. Stokes was the first to propose using absorption, color refraction, and fluorescence in identifying organic substance. When we compare the excited (absorbed) plot to the emission (released energy) plot we can see what is called a “Stokes Shift,” which is vital when looking at data from fluorescence spectrometers. [13]. Fluorescence spectrometers measure the absorbance and emission of the molecule. The emission spectrum shows the molecule when it releases this energy, meaning it will have longer wavelength lower energy. Stokes Shift takes the difference from the absorption peak and the emission peak. From this we can determine more about the molecule; for example, identifying organic and inorganic molecules as well as interactions between the fluorophores and different compounds.

Fluorescence Spectroscopy gives us the visuals we need to interpret these principles of quenching, energy states, etc. Alexander Jablonski is regarded as “the father of fluorescence spectroscopy” because of his many accomplishments in analyzing absorptions and emissions of fluorophores [14]. Jablonski knew that fluorophores emit light at longer wavelengths, but he wanted to understand why this occurred, therefore he made the Jablonski Energy Diagram (Figure 1.2). Jablonski diagrams demonstrate the relationship between electrons and their absorption and emission of light. The diagram depicts the electronic states of a molecule as it changes from one to another electronic state [15]. Fluorescence spectroscopy is being more widely used, mainly because of its high sensitivity. This allows for the detection of molecules at very low concentrations, up to a single atom at a time. The director of the NIH-funded Laboratory for Fluorescence Dynamics at the University of Illinois, Theodore Hazlett, stated “[fluorescence spectroscopy is used due to] its ability to adapt to a multitude of conditions and report on a wide range of effects, which has given it widespread usage" [16]. Fluorescence spectroscopy does not only tell whether a compound fluoresces or not. It can also tell us the interactions between a fluorophore and another substance, such as DNA and RNA. Interactions between fluorophores and genetic material is the basis of cellular imaging, and much government funding [16].

Fluorophores can act as a ligand when bound to genetic material. The ligand is just the smaller molecule bound to a macromolecules, either a protein or DNA. Cresyl violet acetate acts as a ligand when bound to DNA. It is an aromatic and planar dye. It is cationic, with an oxazine ring in its structure. Through studies it was determined to be in its monomeric form at low concentrations but is highly dimerized at high concentrations. There are two observable pKa values, one at 7.91 and the other at 10.77. Because of its oxazine ring it can readily form H- aggregates on negatively charged nanocyrstallites [20].

1.4 Nucleic Acids

Deoxyribonucleic acid (DNA) is the macromolecule that will be used in the proceeding experiments. DNA is the genetic material, which is transcribed into ribonucleic acid (RNA). DNA is composed of nucleotides. Nucleotides contain a phosphate group, pentose sugar, and a nitrogenous base. In DNA the nitrogenous bases are adenine and guanine (purines) and cytosine and thymine (pyrimidines). It forms a double stranded double helix composed of a 5’ to 3’ end strand and 3’ to 5’ end. Adenine and thymine interact through two hydrogen bonds. Guanine and cytosine are stronger because they form three hydrogen bonds between the two. DNA contains deoxyribose sugars missing a hydroxyl group at the 2’ end of the sugar ring. RNA contains the same purines, however, thymine is exchanged for uracil in the pyrimidines. RNA contains a ribose sugar backbone with has a hydroxyl group at 2’ carbon. This makes RNA more unstable than DNA and is more reactive. RNA is also single stranded.

1.5 Modes of Interactions: Intercalation

The interaction between a fluorophore and genetic material is becoming increasingly popular because there has been an increase in research for new efficient drugs targeting DNA for things such as genetic mutations (damage within nucleic acids), anticancer drugs treatment, and as sequence specific binding and cleavage agents [17]. The best way to understand the interaction is to understand it on a molecular level. Ligands can bind to DNA by intercalation, electrostatic interactions, and minor and major groove interactions [18]. Electrostatic interactions include hydrogen bonding, van der waals, and salt bridges. Minor and major groove interactions are bond interactions depending on available space between the base pairs on DNA. Intercalation is when a planar and heteroaromatic molecule is able to slide in between the DNA base pairs [19]. Although intercalation exists, there are not too many DNA-interactive agents that are characterized in atomic detail. Nevertheless, there are several ways in determining whether a molecule is intercalating with DNA or not. This is important because as research grows, now intercalation of 3 dimensional structures is becoming a possibility and therefore 2 dimensional intercalation needs to be well understood.

Intercalative interactions were proposed by L. S. Lerman in 1961. They discovered that during intercalation there is first an unwinding and lengthening of the DNA double helix. This occurs because he base pairs and backbone need to makes space for the intercalator to slide in. Then electronic interactions of the intercalator occur within the helix. What occurs is a π—π stacking between the aromatic planar intercalator molecule and the heterocyclic base pairs. This occurs through dipole-dipole interactions. This causes rigidity with structural overlaps between the π—π stacking. There are several experiments in order to tell intercalation. An experiment that can be done using spectroscopy is where the there can be an indication of the electronic interaction between the intercalator and DNA bases. Binding of intercalators with DNA usually results in hypochromism and shift to longer wavelength of the transition of the intercalated chromophore. Hypochromism is when there is a change in light absorption after the DNA polynucleotides goes from an ordered alpha double helix state, to a disordered, denatured state [21]. There is either emission enhancements or quenching when the intercalator luminesces. There can also be chanced in the excited state electronic structure. An H proton nuclear magnetic resonance (NMR) upfield shift can mean a π—π stacking [19].

1.6 Cresyl Violet

Fluorescence dyes have become increasingly popular in biological research because of its high sensitivity and selectivity. Because of the use of fluorescence dyes, more are being analyzed and created for biological experiments. Cresyl violet acetate (C18H15N3O3) is a small organic dye molecule that is aromatic and planar. Aromaticity is the extra stability possessed by a molecule where the pi bonds all lie within a cyclic structure. There is a loop of p orbitals, where p orbitals must be planar and overlap, which following Huckel’s Rule of 4n+2 electrons in the delocalized p-orbital cloud [23]. Cresyl violet is most commonly known for being used in two disciplines: first as a stain of neuronal tissues, and secondly as a standard for red-region emission in fluorescence experiments. Cresyl violet acetate is fairly high in solubility. It is also an oxazine dye, where it has a ring with nitrogen and oxygen in the para position. Cresyl violet is a monomer at low concentrations ranging lower then 20 uM. It is a dimer as concentrations higher then 20 uM. Dimers are common in aromatic compounds due to the ability to stack because of pi orbitals. Cresyl violet is also cationic, with a positive charge on one of its amine groups NH2+. Because of the cationic character, at different pHs the structure of cresyl violet may change. When that positive charge exist it can drive reactions or attractions both to itself as well as other molecules, such as DNA. Cresyl violet has two observable pKa values: pKa1: 7.91, pKa2: 10.77. The pKa, is -log(Ka) where Ka is the acid dissociation constant, which determines the strength of the acid in solution. On a pH scale, pH of 7 is neutral, below that is acidic, above that is basic. The pH express the concentration of hydrogen’s in solution (or H3O+) A basic solution is a solution capable of accepting a proton from an acid. The pH and pKa are related through the Henderson Hasselbalch equation. There are two forms of the equation.

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An example of an acid base titration, to show the effects of pH with acid and bases, and where to locate equivalence points and where the pH = pKa. Buffers rely heavily on pKas and solutions that will neutralize with excess acidity, or excess alkalinity. This is why buffers are usually weak acids or weak bases because of their titration curves. There is less pH change in weak acid/weak base titrations, rather than strong acid strong base titrations. Buffers are used because their resistance to change in pH. Cresyl violet acetate is said to have an excitation wavelength between 540-585 nm [22]. Knowing the structure of cresyl violet and the structure of DNA, spectroscopy can be used to predict modes of interaction.

1.7 Scatchard Plot Analysis

Scatchard plots determine ligand binding using spectroscopy. In biochemistry, fluorescence spectroscopy can be used to create scatchard plots. A Scatchard plot of the binding data is then calculated by plotting the ratio of bound/free ligand versus the bound fraction and calculating the slope [26]. In order to do this, the fluorescence maximum of all ligands bound to DNA must be known. The data acquired is using a single cuvette. The concentration of a free ligand does not need to be known, only the concentration of bound and unbound ligand is needed. There is a modified scatchard plot that involves the reciprocal plot of [DNA]/f versus 1/(1-f). The slope yields the binding constant Kf and the intercept gives the value N. N is the value for the number of base pairs involved in binding a ligand [26]. The binding affinity, Kf, of ligand, L, and macromolecule, S, and its formed complex, SL, is given in equation 9.

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[SL] is the number of occupied macromolecule sites, and [S] is the number of unoccupied macromolecule sites, and [L] is free ligand. If Nocc represents the average number of occupied sites of DNA per ligand, and N represents the total number of binding sites then the ratio of Nocc and N can be written as,

A plot of this data would give a hyperbolic curve, which is hard to analyze, therefore it is rearranged to give a linear graph. To do this, the term f is determined using Fobs and Fmax, and FL.

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Then [SL] can be substituted using equation 9.

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A plot of this data would give a hyperbolic curve, which is hard to analyze, therefore it is rearranged to give a linear graph. To do this, the term f is determined using Fobs and Fmax, and FL.

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Where Fobs is the fluorescence observed after the addition of DNA. Fmax is the maximum absorbance when all ligand is bound, and FL is fluorescence of free ligand.

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Equation 13 represents the total concentration of bound and unbound ligand and concentration of the macromolecule. Therefore equation 11 and 13 can be set equal to each other

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It can then be rearranged as

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Then 1-f represents the fractions of free ligand can be seen as,

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Therefore,

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Where [L]totai is the initial concentration of fluorescence dye. [S]totai is the concentration of DNA with each titration. Then a plot of 1/(1-f) versus [illustration not visible in this excerpt] will give a slope of 1/NKf and an intercept of [illustration not visible in this excerpt] N represents the number of ligand binding sites per nucleotide. 1/N is a more reasonable value, which give the number of nucleotides per ligand [26].

1.8 Computer Modeling: Molecular Operating Environment

In a time of technological advancements, computer modeling has become increasingly popular in detecting interactions between ligands and macromolecules. The Chemical Computing Group designed a computational chemistry program called Molecular Operating Environment. It is a drug discovery software package capable of structure-based design, fragment based design, pharmacophore design, protein and antibody applications, and importantly molecular modeling and simulations. The main focus of the program is to be able to predict the modes of interactions between cresyl violet, the ligand, and DNA, the macromolecule. Therefore we need to make the models for these structures and perform simulations. Simulations allow scientist and researchers to validate ligand stability and geometries. The program has built in operations to determine the best geometry of the molecules, where there are missing disulfide bridges of proteins, the charges of the molecules, or empty residuals to optimize the structure. The program is built upon molecular mechanics, or force fields. Force fields use basic arithmetic in attempt to provide the most stable potential energy surface in the different arrangement of atoms in space of a molecule. Force fields characterize structure and movement as potential energies. Therefore the actual energy values given in the program have no real significance, because it is a hypothetical value. However, E is an arbitrary value that shows how much steric energy there is within the molecules. It is a combination of energy for bond stretching, bond angle bending, torsional energy, non-bonded interactions, and electrostatic repulsion [24]. The MOE program provides standard molecular mechanics force fields AMBER 94/99, CHARMM 27, MMFF94(s), OPLS-AA and Engh-Huber that run on multiple CPU threads [25].

When docking is performed there will be a number of ligand configurations, called poses that are given and scored. Scores are the free energy of binding. The poses with the lowest scores are considered the best poses. This is conformational analysis, where we have different docking methodologies to apply the correct torsion angles, rotatable bonds, bond lengths, and bond angles, as well as having rings flex or not flex. Placement is used to help score poses generated from pool of ligand conformations. Rescoring generates poses by generating scoring functions to emphasize favorable hydrophobic, ionic, and hydrogen bonding contact. There are several rescoring parameters; ASE scoring, Affinity dG, Alpha HB, and London dG. ASE scoring takes the sum of Gaussians over all ligand atom-receptor atom pairs and ligand atoms. Affinity dG estimates enthalpic contributions of free energy of binding using a linear function. Alpha HB basis off of hydrogen bonding and geometric fit of ligand binding. London dG estimates free energy of binding of a ligand from a given pose. Refinement generates poses based on forcefields or Gridmin. Forcefield is more accurate than GridMin parameters because GridMin is spatial arrangements, where force fields are based off of mathematical functions used to describe potential energy as a system of particles. The docking parameter’s that produce the lowest score and give consistent results is selected as the most reasonable ligand-receptor binding mode. The docking output data poses show the following values; Mol—an output pose, S—final score given to the pose given in kcal/mol, Rmsd_refine—root mean square deviation between the pose before and after refinement, E_conf—energy of the conformer, E_place—score from the placement state, E_score—score from rescoring stage, and E_refine—score from refinement stage [27].

Through spectroscopy and computer modeling researchers and scientist can determine a large amount of information about different molecules from organic substances, to inorganic substances. Fluorescence dyes in the recent years are becoming increasingly popular in biological studies, but in order to use fluorescent dyes in biological systems, spectroscopy and computer modeling are useful tools to see if they are practical substances to use. Cresyl violet when bound to DNA, it strains the DNA blue. However, when bound to ribonucleic acid (RNA) is stains purple (Figure 1.4). Then using single stranded DNA of 15’poly A, 15’poly T, 15’poly G, and 15’poly C, we see that depending on the base pair there is a difference in color from blue and purple as well (Figure 1.5). This phenomenon is hypothesized that there is a change in the conjugation system of cresyl violet when bound to the genetic material. However, it is through spectroscopy and computer modeling that we hope to prove what modes of interaction are occurring within this system.

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Figure 1. 4 Gel Electrophoresis of DNA and RNA (Done by Stephanie Chapelliquen)

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Figure 1. 5 Gel Elextrophoresis of 15'Poly ssDNA semengts of DNA of different bases and Chimera . . . . strand of alternating dna and RNA nucleotides there is a change in the conjugation system of cresyl (Done by Stephanie Chapelliquen)

Chapter 2: Experimental

The instruments used were the F-2500 Fluorescence Spectrophotometer, Hitachi, AJN Scientific Inc. with the FL solutions program, the Agilent Technologies 845x UV Visible System, and the Molecular Operating Environment (MOE) program from Chemical Computing Company. Chemicals and materials used were DNA calf thymus ordered from Sigma-Aldrich with no more purification (CAS No. 91080-16-9), 15 base pair single stranded DNA of guanine, adenine, cytosine, and thymine, from Integrated DNA Technologies, cresyl violet acetate ordered from Sigma-Aldrich (CAS No. 10510-54-0), tris(hydroxymethyl)-aminomethane from Sigma- Aldrich (CAS No. 77-86-1), sodium chloride from Acros (CAS No. 7647-14-5), hydrochloric acid, in house (CAS No. 7647-01-0), sodium hydroxide, in house (CAS No. 1310-73-2), spermidine ordered from Sigma-Aldrich (Lot # BCBH7695V, CAS No. 124-20-9), and γ- cyclodextrin ordered from Sigma-Aldrich (Lot #5LBB4943V, CAS No. 17465-86-0).

2.1 Computer Modeling

The Molecular Operating Environment (MOE) program was used to do docking experiments. First the molecules need to be constructed, both the DNA and cresyl violet molecule. Cresyl violet molecule (figure 2.4) was constructed using a construction tutorial provided by МОЕ. It is important to have the lowest and most Figure 2. 1 Cresyl Violet Acetate Molecule stable potential energy for the molecules that will be docked. To check stability of the energy, select WINDOW, POTENTIAL SETUP. Then there will be parameters that can change. For cresyl violet the parameters need to be force field, MMFF94X, and solvation changes to gas phase, then press ok. The reason force field is MMFF94X is because cresyl violet is a small molecule. Then go to Giz-MOE, ENERGY, and the energy of the molecule will show at the top left corner of the screen. To convert the energy to the lowest possible energy, use the MINIMIZE icon on the right hand side of MOE. At the lowest possible energy cresyl violet is 72.3190 kcal/mol. This is a sum of all the different energies MOE accounts for. To find out the breakdown of the energies select, COMPUTE, POTENTIAL ENERGY, and all the energy breakdowns will show at the top of the screen. A way to make sure that MOE calculated sufficient data is that one of the energies is the out of plane bending (opp), because cresyl violet is a planar molecule it should be an opp of 0.000kcal/mol, which there is. This molecule was saved in the protein database file (pdb) so that the molecule did not need to be constructed for every docking we performed [27].

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Figure 2. 1 Cresyl Violet Acetate Molecule

Then construction of DNA both single stranded and double stranded DNA was done. MOE already has the structures of genetic material and proteins built in. To construct double stranded DNA we go to EDIT, BUILD, SEQUENCE. From there we can enter the required sequence for each docking experiment. The base pairs are A-adenine, T-thymine, G-guanine, C- cytosine, and U-uracil. To construct single stranded DNA simply select the one stand not desired and delete the entire row. Then, as with the cresyl violet molecule, once the molecule is constructed the potential energy is calculated by selecting first selecting the correct force field. Depending on the size of the DNA structure, either MMFF94X or CHARMM 27 will be used. MMFF94X is used for smaller, single stranded DNA. CHARMM 27 is used for larger, double stranded DNA. Therefore the lowest potential energy needs to be determined by running a energy minimization with each DNA construction.

To perform docking use the docking panel, MOE, COMPUTE, SIMULATIONS, and DOCK. In OUTPUT the filename is written, cresyl violet molecule selected as LIGAND, and the DNA strand is selected for RECEPTOR. In SITE, select the receptor. PLACEMENT will change with each experiment as with RESCORING. REFINEMENT stays as force field, and the second RESCORING will vary. The combination of docking methodologies with the lowest and most consistent potential energy will provide a general input of what type of ligand-receptor interaction is occurring.

2.2 Fluorescence Analysis

Fluorescence spectroscopy was used to run concentration control experiments, and to see that change in emission due to pH and titrations of different DNA fragments. All experiments were done at room temperature. First, it is important to know how to run a fluorescence scan using the F-2500 Fluorescence Spectrophotometer. A pre-scan is used to get a rough estimate of the emission and excitation wavelengths, which are needed when running an emission and an excitation spectrum scan. These wavelengths, given in nanometers (nm) are used when creating a method. Using a quartz cuvette, the sample is placed into the spectrometer. Then using the FL Solutions program, select METHOD on the tool bar located on the right-hand side of the screen, select under scan mode EXCITATION, then select OK. On the tool bar on the right-handed side, select PRE-SCAN. In the upper right corner, numbers will be increasing and decreasing in the block labeled EX WL. This number is the EX WL is use when running an emission spectrum. Then select METHOD on the tool bar located on the right-hand side of the screen, select EMISSION under scan mode, and select OK. Select pre-scan and then in the upper right corner, there will be values increasing and decreasing in the EM WL block. This value is used when running excitation spectrums. For cresyl violet acetate, the EM WL is 620 nm and EX WL is 585 nm. To run an excitation fluorescence spectrum, METHOD is selected from the tool bar, then the instrumental tab is selected to set up the method. Method is as followed:

- Scan Mode: Excitation
- Data Mode: Fluorescence
- EM WL (emission wavelength): enter the emission wavelength
- EX Start WL (excitation start wavelength): enter # (usually 200 nm)
- EX End WL (excitation end wave length): enter # (usually 800nm)
- EX Slit: 2.5 or 5.0 (either displays a good spectrum)
- EM Slit: 2.5 or 5.0 (either displays a good spectrum)

Once the method is set up, OK is selected. The sample is placed in the spectrometer, MEASURE is selected from the tool bar. Once the spectrum is complete, it will appear in a second window. The maximum peaks are reported, and a report of the spectrum can be saved. To run an emission fluorescence spectrum the scan mode needs to be changed to EMISSION, and the EX WL is changed to the EX WL given from the pre-scan (Credit to Brianna Hill, Fairleigh Dickinson University).

2.3 UV-Visible Spectroscopy Analysis

UV-visible spectroscopy was used to monitor the monomer and dimer ratios of cresyl violet when under different conditions. The Agilent Technologies 845x UV Visible System was used, the program was simple to use. The blank was placed in a UV-visible cuvette and the blank button was selected on the actual instrument. Then the sample was placed in the UV-visible cuvette and placed in the instrument and the sample button was selected on the actual instrument. The spectrum was set to scan from 300 - 800 nm. Peaks were observed at 562 nm (monomer) and 585 nm (dimer).

2.4 pH Analysis

To prepare the solutions to be tested using fluorescence spectroscopy, a stock solution of 53 uM cresyl violet was prepared dissolving in distilled water. Then there was a half dilution, fourth dilution, eight dilution, and a sixteenth dilution made from the stock. Each diluted sample was then separated to three batches and pH was adjusted to 5, 7, and 8 using 0.1% hydrochloric acid and 0.05N sodium hydroxide. Each sample was ran through an emission scan using the F- 2500 Hitachi Fluorescence Spectrophotometer and absorption scan using the Agilent Technologies 845x UV Visible System. Since there were serial dilutions, a concentration analysis was done as well.

2.5 Scatchard Plot Analysis

Based off of the experiment done by Williams and Dsilva (2011) a 250 mL stock solution of a 5mM tris, 50mM sodium chloride buffer of pH 7.2 (tris buffer) was made for the DNA and cresyl violet solution. All IDT ssDNA samples were prepared by dissolving in 1mg/1mL solution of buffer mention above. Calf thymus DNA was dissolved in 1mg/1mL solution of 50 μΜ sodium chloride solution. The DNA concentration of calf thymus was determined by measuring the absorbance at 260 nm and using the equation provided by Sigma-Aldrich ^g/mL of DNA = A260*50μg/mL * Dilution Factor). The stock concentration of DNA was 0.5mM calf thymus. For the IDT ssDNA concentrations were determined using the Oligo I and Oligo II concentration after measuring their absorbance at 260 nm. Guanine concentration was 205.1 μM, thymine 222.2 μM, adenine 215.7 μM, and cytosine was 233.9 μM as their stock samples.

Then 3-mL solution of ^M of cresyl violet solution was prepared in tris buffer solution. Fluorescence emission scans were run after each addition of DNA. The concentrations were selected so that there were fluorescence scans of samples where cresyl violet concentration was higher than the DNA concentration, then where they were a 1:1 ratio, and then when cresyl violet was saturated with DNA. Each titration consisted of roughly 10-15 different samples adding 2 - 5 μΜ of DNA during each sample. The cuvette was mixed after each addition of DNA for roughly 30 seconds. Fluorescence value at 620 nM was recorded after each sample scan, and then later analyzed using scatchard plot method.

Chapter 3: Results and Discussion 3.1 Computer Modeling

The Molecular Operating Environment (MOE) software developed by Chemical Computing Group as a fully integrated drug discovery package was used for docking of cresyl violet with varying DNA fragments. The first important parameter to set is the force field. Of the three parameters (MMFF94X, CHARMM27, and AMBER99) using 15’ Poly C ssDNA, MMFF94X gave the lowest potential energy of -3010.7961 kcal/mol. CHARMM27 gave a potential energy of -1244.4718 kcal/mol, and AMBER99 gave a potential energy of -2745.8806 kcal/mol. Therefore, for all 15’ poly ssDNA that were built used the MMFF94X as the force field and the energies were consistent (table 1).

Table 3.1 Potential Energies

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Docking of thymine was done previously using MMFF94X force field and the ASE rescoring function produced the lowest scored pose. This pose showed a bond length between the nitrogen of cresyl violet and the nitrogen 1 of thymine to be 4.05, the oxygen of cresyl violet and nitrogen 1 for another thymine gave 4.75, and the positive amine of the cresyl violet and the oxygen of the 3’ ribose sugar gave 3.21 bond length (Figure 3.1). These were considered possible hydrogen bonding occurring between the cresyl violet molecule and DNA helix this is because nitrogen I of thymine has a change of donating a hydrogen and form a hydrogen bond. However, these parameters did not include the FIXED HYDROGENS parameter. The fixed hydrogens parameter states that is there are missing hydrogens or if the force field requires united atoms (implicit hydrogen’s) on some atoms then the fix hydrogens button should be used to add and/or delete hydrogens according to force field specifications [25].

When using the FIXED HYDROGENS parameter, the potential energy decreased from -3088.8432 kcal/mol to -3610.9359 kcal/mol for the 15’Poly thymine ssDNA. Therefore docking was done using the parameters MMFF94X, fixed hydrogens, Triangle Matcher, Affinity dG, force field, Alpha HB. These gave the lowest poses. The minimized energy was -3875.5064 kcal/mol. The trial was done three times, and the energies were not consistent changing from - 3875.5064 to -3606.9783 kcal/mol. However, with each docking there was an interaction between the NH2+ and the oxygen of the phosphate group on the DNA. There were also possible interactions between the oxazine ring of cresyl violet at the thymine ring, as well as the NH2 with oxygen of the thymine base (Figure 3.1). The docking shown in Figure 3.2 was the pose what gave the lowest S value (Figure 3.3).

For a 15 base single stranded DNA structure composed of adenine several docking experiment were done using the parameters MMFF94X, fixed hydrogens, Triangle Matcher, London dG, force field, and then changing the second rescoring. The docking that gave the lowest potential energy was using ASE for the second rescoring. This gave the lowest S scoring, and its lowest S pose was analyzed (Figure 3.4 and 3.6). In Figure 3.6, there is a ligand interaction feature showing the predicted modes of interaction between ligand and receptor (legend shown in Figure 3.5). It shows that there are hydrogens donated from the adenine base to arene rings of cresyl violet. These arene interactions could suggest possible intercalation, however, when looking at the 3D structure in Figure 3.4 we see that it is laying along the side of the DNA structure, rather than in between two bases.

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Figure 3.1 MOE Docking of 15’Poly Thymine with Cresyl Violet molecule without Fixed Hydrogen Method.

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Figure 3.2 MOE Docking of 15’Poly Thymine with Cresyl Violet molecule, showing bond length distances.

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Figure 3.3 The output data poses for MOE Docking of 15’Poly Thymine with Cresyl Violet molecule.

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Figure 3.4 MOE Docking of 15’Poly Adenine with Cresyl Violet molecule.

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Figure 3.5. MOE Docking Ligand Interaction Legend

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Figure 3.6 MOE docking Ligand Interaction of 15’ Poly ssDNA of Adenine

For a 15 base single stranded DNA structure composed of cytosine several docking experiments done using the same procedure as adenine. The docking that gave the lowest potential energy was using Alpha HB for the second rescoring. This gave the lowest S score of -52.5079 kcal/mol, and its lowest S pose was analyzed (Figure 3.6). A surface map feature was added to the Figure to show how cresyl violet embeds itself into the ssDNA of 15’poly cytosine. Both the -NH2 and -NH2+ amine groups interact with the DNA. The -NH2+ interacts with the negative phosphate backbone. The -NH2 of the cresyl violet has a hydrogen donor to the cytosine ring.

When docking cresyl violet with 15’poly ssDNA of guanine, we made sure to add an adenine base every three guanine bases to prevent guanine from coiling upon itself. Once this structure was made, we found the best parameters to be using Alpha HB as the rescoring 2. The S score was -76.5203 kcal/mol. Its lowest S pose was analyzed (Figure 3.8 and 3.9). This interaction also showed an interaction between the amine groups and the negative phosphate backbone.

After analyzing all the 15’poly ssDNA docked with cresyl violet, there was a prominent interaction between the negative phosphate backbone and the positive charged amine group of the cresyl violet. This information, gives us insight that there is electrostatic interactions between cresyl violet and 15’poly ssDNA. However, this would not account for color changes. The reason being is that there is no change in the conjugation of the cresyl violet. Conjugation is what accounts for color changes. There was also no intercalation (π to π bonding). There was an H- arene interaction detected with the 15’poly ssDNA of adenine, however, intercalation would be an arene-arene interaction.

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Figure 3.7 MOE docking of 15’ Poly Cytosine ssDNA

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Figure 3.8. MOE docking Ligand Interaction of 15’ Poly ssDNA of Guanine

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Figure 3.9 MOE docking of 15’ Poly Guanine ssDNA

The next sets of experiments were done to see the effects of pH on cresyl violet bonding with double stranded DNA. Cresyl violet molecule was built and saved with a 1+ charge, neutral charge, and 2+ charges. In basic solution the -NH2+ will become -NH. When docking this to dsDNA with the sequence 5’CAT TAG GAG TAT AAT 3’, the best parameters were using Alpha HB as the rescoring 2. This showed a backbone interaction with the -NH2 amine group with a 27% change of a hydrogen bond, electrostatic interaction with a thymine base (Figure 3.10 and Figure 3.11). In neutral pH solution, the cresyl violet molecule will be with its cationic character and have a +1 charge. Docking using the Alpha HB rescoring parameter showed two backbone interactions at both amine ends. The interactions were electrostatic. The NH2+ electrostatically interacted with an adenine base and the -NH2 electrostatically interacted with a thymine base (Figure 3.12 and 3.13). These interactions involved two possible hydrogen bonding sites and non-hydrogen bonding interaction between -NH2 and the O' of the phosphate group of the DNA backbone. Lastly, cresyl violet molecule with a +2 charge was docked with dsDNA and there was also two interactions. The interactions were electrostatic. The NH2+ electrostatically interacted with an adenine base and the -NH3+ electrostatically interacted with a thymine base (Figure 3.14 and 3.15).

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Figure 3.10 MOE docking Ligand Interaction of 15’ Poly dsDNA with Neutral Charge Cresyl Violet

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Figure 3.11 MOE docking of 15’ Poly dsDNA with Neutral Charge Cresyl Violet

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Figure 3.12 MOE Ligand Interaction of 15’ Poly dsDNA with +1 Charge Cresyl Violet

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Figure 3.13 MOE Docking of 15’ Poly dsDNA with +1 Charge Cresyl Violet

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Figure 3.14 MOE Ligand Interaction of 15’ Poly dsDNA with +2 Charge Cresyl Violet

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Figure 3.15 MOE docking of 15’ Poly dsDNA with +2 Charge Cresyl Violet

3.2 pH experiments

All solutions were made in deionized water, and their pHs were adjusted with hydrochloric acid and sodium hydroxide. The excitation wavelength (EX WL) was 585 nm, and the emission wavelength (EM WL) was 620 nm, due to Rayleigh scattering of light particles there are peaks corresponding to the EX WL and EM WL present in every spectrum. We had four different concentrations 3.33, 6.65, 13.40, and 26.4 uM. We then took each concentrated solution and made a sample of pH 5.0, 7.0, and 8.10. We ran excitation and emission spectrums of each to see if there were any variations of the peaks. There were no shifts in peaks, only changes in intensity. Fluorescence excitation and emission spectroscopy showed a decrease of intensity at in the presence of basic solution (sodium hydroxide) and increase of intensity in acidic solution (hydrochloric acid) (Figure 3.16 and 3.17). The excitation spectra showed a decrease in both monomer (562 nm) and dimer (585 nm) peak. This could be due to the proton transfer from the -NH2 of cresyl violet to the -OH", to make a doubly charged species of cresyl violet (deprotonation). The increase in fluorescence due to hydrochloric acid could be due to the protonation of the cationic amine group of cresyl violet. At the pH’s selected there are still the monomer and dimer formation of the cationic (+1) charged cresyl violet, we assume that there is no shift in peaks unless at extreme pHs (pH of 1 to pH of 14). Therefore pH of 5 had the highest intensity, meaning the most amount of the +1 cationic charged cresyl violet, with a mixture of +2 charged cresyl violet. However, because there was not a large increase in intensity of cresyl violet fluorescence between pH of 5 and pH of 7, it was decided that a pH buffer of 7 was most ideal for scatchard plot analysis, since the pH range in our biological systems are between 7.3 and 7.4 we would see binding sites in that range because we hope to use cresyl violet as a

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Figure 3.16 Fluorescence Excitation Spectrum for 26.5 uM cresyl violet

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Figure 3.17 Fluorescence Emission Spectrum for 26.5 uM cresyl violet

biological tracer. An overlay of all the spectra for varying pH and concentrations are show in Figure 3.18 and 3.19.

Fluorescence excitation spectrum can also determine monomer to dimer ratio with increasing or decreasing concentration of cresyl violet (Figure 3.20). The monomer to dimer ratio is also very important when comparing to docking, the monomer peak is at 562 nm and the dimer is at 620 nm. With a concentration of 26.5 uM cresyl violet there is much more dimerization present than monomers. However, at low concentrations more monomer is present. Fluorescence emission spectrum can show us relative concentrations based off fluorescence intensity as well (Figure 3.21). With increasing concentration, there is an increase in fluorescence intensity.

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Figure 3.18 Overlay of all Excitation Spectra (varying pH and varying concentration)

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Figure 3.19 Overlay of all Emission Spectra (varying pH and varying concentration)

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Figure 3.20 1564/Ιβ22 vs. Concentration of Cresyl violet

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Figure 3.21 Fluorescence Intensity vs. Concentration (EX WL 585 nm) pH of 5.02

3.3 Fluorescence Effects of Cresyl Violet Bound to DNA

Fluorescence intensities are affected when cresyl violet is bound to DNA. There were five different strands of DNA that were analyzed. Cresyl violet, at a concentration of 4 uM in tris buffer solution was mixed with 4uM of each strand of DNA (15’ Poly ssDNA of all Adenine, Thymine, and Cytosine, 15’ Poly ssDNA of guanine 5’ GGG GAG GGG GAG GGG 3’, and lastly dsDNA calf thymus). It was observed that there are shifts in the original 620 nm peak of cresyl violet, with no DNA present (Figure 3.22). Cresyl violet when bound to thymine has a red shift of 6 nm. When bound to cytosine there is an 11 nm red shift. Adenine had a red shift of 9 nm, and calf thymus and guanine shifts were undetectable because of its quenching. It was also observed that there is quenching of cresyl violet when bound to DNA. This means that there was a decrease in fluorescence intensity. The Stokes Shift would be due to possible hydrogen bonding between cresyl violet and DNA. The quenching is due to an electron sharing or donor properties of the adjacent nitrogenous base next the fluorophore (cresyl violet). The order of quenching efficiency is G<A<C<T if the nucleobase is reduced but it is the reverse, G>A>C>T, if the nucleobase is oxidized [29]. We see this in our spectra, because guanine quenches almost all the fluorescence of cresyl violet, to the point where the spectral shift cannot be detected. This also shows that this hydrogen donating prediction from docking is very possible between nitrogenous bases and cresyl violet at the amine groups.

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Figure 3.22 Fluorescence Emission Spectrum of Cresyl Violet when bound to different DNA strands

3.4 Scatchard Plot

Scatchard Plot analyses were done after pH was determined best at neutral conditions, mainly because of its maximum fluorescence intensity. Since DNA acts as a fluorescence quencher the fluorescence intensity decreases with increasing amount of DNA added (Figure 3.23). In Figure 3.23, we see the titration of dsDNA calf thymus in 1uM cresyl violet, and the fluorescence intensity decreases as concentration of DNA increases. We did a scatchard plot analysis comparison with varying concentrations of cresyl violet (Figure 3.24). The 1/N value and pKb were calculated and given in table 2. When observing 1/N value versus concentration we see that at very low concentrations on cresyl violet there is less binding and at very high concentrations there is less binding, however at 25 uM there was more binding. This could be because at low concentrations cresyl violet, there is less free ligand to bind to the large calf thymus DNA strands, and at very high concentrations there is too much dimerization inhibiting binding.

Table 3.2 Scatchard Analysis Data for Varying Concentration Cresyl Violet

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Our focus is to try and determine if intercalation is a mode of interaction between cresyl violet and DNA. This is because intercalation is very important in genetic mutations. Docking showed backbone interactions and scatchard analysis based on concentrations shows that at high concentrations dimerization inhibit binding. Therefore, spermidine (Figure 3.26) was used to inhibit electrostatic interaction with the backbone of DNA, and γ -cyclodextrin (Figure 3.27) was used to inhibit dimerization of cresyl violet.

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Figure 3.23 Fluorescence Intensity vs. Wavelength of 1uM cresyl violet with varying concentrations of DNA

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Figure 3.24 Scatchard Plot Analysis of dsDNA calf thymus as varying concentrations of Cresyl Violet

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Figure 3.25 1/N value vs. Concentration of Cresyl Violet

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Figure 3.26 Spermidine Structure

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Figure 3.27 γ-Cyclodextrin Structure

Experiments were performed with γ -cyclodextrin and cresyl violet to prove that it inhibits dimerization. However, it does not inhibit dimerization, the dimer actually sits inside the γ-cyclodextrin structure and becomes non-fluorescent. Using 1 uM cresyl violet, varying concentrations of γ-cyclodetrin was added, and the absorption spectra was taken using an UV- Visible spectrophotometer (Figure 3.28). Then using Peak Fit v4.12 software the peak composition of each spectrum was obtained. Each spectrum was broken down into two major peaks, the monomer peak at roughly 562 nm, and the dimer peak at 585 nm. The R2 values were all near 0.996. An example peak fit spectra is shown in Figure 3.29. Then the height of each monomer peak and dimer peak were recorded and a ratio of Monomer/Dimer was done and showed that at 40 uM γ-cyclodextrin has a monomer dimer ratio of 0.85 and 600 uM γ- cyclodextrin has a monomer dimer ratio of 1.20. The peak heights were also compared in a bar graph representation to show, that although dimers are always present, γ-cyclodextrin does in fact help decrease dimerization, but in doing so there is also some decrease in absorbance (Figure 3.30).

Scatchard analysis was then done to see if there would be more binding when inhibiting backbone electrostatic interactions, when inhibiting dimerization, and then when inhibiting both at the same time. A fluorescence titration was done for each sample adding small increments of dsDNA calf thymus and our 1/N values and pKb values were calculated and give in table 3. It showed that electrostatic interactions and dimerization to inhibit cresyl violet from interaction with the nucleotide base pairs because we see that as we in inhibit these interactions, we see more ligands binding per base pair, since 1/N value means one ligand bound every 1/N times. The scatchard plots were overlaid to see the change in slope with each sample (Figure 3.31).

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Figure 3.28 The Absorption Spectra of Cresyl Violet with varying concentrations of γ-Cyclodextrin

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Figure 3.29 The Peak Fit Analysis of 1uM Cresyl Violet with 40 uM γ-Cyclodextrin

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Figure 3.30 Concentration of y-cyclodextrin vs. Absorbance at 562nm and 585 nm

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Figure 3.31 Scatchard Plot Analysis using y-Cyclodextrin and Spermidine

Table 3.3 Scatchard Analysis Data using Spermidine and y-Cyclodextrin

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Through scatchard plots, it was seen that concentration, backbone interactions, and presence of dimers affect binding. The best binding was with 1 uM cresyl violet solution, 600 uM γ -cyclodextrin, and 1.0e-04 M spermidine. This showed that for every 10 nitrogenous bases there was one ligand bound. The mode of this interaction is still not very well understood. It is suggested through docking and fluorescence spectra shifts, that there are hydrogen bonds being formed between the DNA and cresyl violet. Guanine is a strong quencher upon fluorescence because of its ability to donate an electron, therefore is can donate its electron to cresyl violet, decreasing the fluorescence.

The red shifts and hypochromism of the fluorescence spectrums (Figure 3.22) do suggest that hydrogen bonds and possible intercalation are modes of interactions. Scatchard analysis have binding constants that can suggest intercalation, however, the n values are low for intercalation. Typically values of n between 3-4 suggest intercalation, however, the values for these scatchard plots were > 1.00. Intercalation have high n values because there is π-π stacking, meaning multiple binding sits for just the 4 bases it intercalates with.

By doing these experiments it is shown that DNA and cresyl violet do interact in some way, and that hydrogen bonding is present. Intercalation is strongly suggested through the shift and quenching of fluorescence. Nevertheless more experiments doing scatchard plots with different strands of DNA, both double stranded and single stranded can conclude more information. Fluorescence Resonance Electron Transfer experiments, and time-resolved experiments could be done to determine bond lengths. Proton NMR as well as IR could also show any changes within the cresyl violet structure, which could predict changes in the conjugated system. A change in the conjugate system could give insight to the color differences. If we are able to determine binding sites depending on base pairs, and if there is a presence of interalaction, we could then do furture experiments using cresyl violet as a DNA tracer. Cresyl violet could also be possibly used for drug discoveries for genetic mutations.

Reference:

[1] Zhang, Guomei, Shaomin Shuang, Chuan Dong, Diansheng Liu, etc. (2004). Investigation on DNA assembly to neutral red-cyclodextrin complex by molecular spectroscopy. J Photochem PhotobiolB. 27(2-3):127-34. Print. Retrieved on January 5, 2012 from http://www.ncbi.nlm.nih.gov/pubmed/15157908

[2] Introduction to Spectroscopy. (2013). Michigan State University. Retrieved on March 10, 2013 from http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/ Spectrpy/spectro.htm

[3] Spectroscopy Types. (2012). News Medical. Retrieved on March 10, 2013 from http://www.news-medical.net/health/Spectroscopy-Types.aspx

[4] Roman M. Balabin, Ravilya Z. Safieva, and Ekaterina I. Lomakina (2007). "Comparison of linear and nonlinear calibration models based on near infrared (NIR) spectroscopy data for gasoline properties prediction". Chemometr IntellLab 88 (2): 183-188. doi:10.1016/j.chemolab.2007.04.006.

[5] Raman Spectroscopy Basics. (2012). Princeton Instruments. Retrieved March 15, 2013 from http://content.piacton.com/Uploads/Princeton/Documents/Library/UpdatedLibrary/Rama n_Spectroscopy_Basics.pdf

[6] Skoog, Holler, Crouch. (2013). Instrumental Analysis. Cengage Learning. Print.

[7] McMurry, John. (2011). Organic Chemistry, 8th edition. Brooks Cole. Print.

[8] Kafafi, Zakya H. Journal of Photonics for Energy. SPIE. Retrieved March 1, 2013 from http://spie.org/Images/Graphics/Publications/PM105_Fig1.1jpg

[9] Harris, 2001

[10] The Uncertainty Principle. (2009). Georgia State University. Retrieved February 13, 2013 from http://hyperphysics.phy-astr.gsu.edu/Hbase/uncer.html

[11] Davidson, Michael W. (2013). Jablonski Chart. Florida State University. Retrieved February 23, 2013 from http://micro.magnet.fsu.edu/optics/timeline /people/antiqueimages/jablonskifigure1jpg

[12] Spectroscopy. (2010). Intech—Open Access Company. Retrieved on February 23, 2013 from http://www.intechopen.com/source/html/18835/media/image2.jpeg

[13] Fluorescence Fundamentals. (2011). Invitrogen.com. Retrieved from http://www. invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Intro duction -to-Fluorescence-Techniques.html#head1

[14] Lakowicz, Joseph R. (2006). The Principles of Fluorescence Spectroscopy. Baltimore, MD: Springer Science+ Business Media, LLC.

[15] Davidson, Michael W. (2009). Alexander Jablonski, PhD. Fluorescence Spectroscopy. LuminariesLABMedicine, 40:11, 694-695. Retrieved December 5, 2011, from http://labmed.ascpjournals.org/content/40/11/694.full.pdf+html

[16] Fluorescence Research Grows on All Fronts. (2005). Advantage Business Media. Retrieved from http://www.rdmag.com/Featured-Articles/2005/03/Fluorescence-Research-Grows- on-All-Fronts/

[17] Coury, Joseph E., Lori McFail-Isom, Loren Dean Williams, and Laurence A. Bottomley. (1996). A Novel Assay for Drug-DNA binding mode, affinity, and exclusion number: Scanning force microscopy. Proc. Natl. Acad. Sci. 93, 12283-12286. Retrieved February 19, 2013, from www.pnas.org/content/93/22/12283.full.pdf

[18] Song, Jin-Ping, Yu-Jing Guo, Qiang Zhao, Shao-Min Shuang, Chaun Dong, and Martin M.F. Choi. (2010). Assemblies of brilliant cresyl violet to DNA in the presence of γ- cyclodextrin. Talanta. 82, 681-686. Retrieved January 27, 2010, from http://www.elsevier.com/locate/talanta

[19] Long Eric C. and Jacqueline K. Barton. (1990). On Demonstrating DNA Intercalation. Accounts of Chemical Research. 23:9, 271-273. Print.

[20] Di Liu, Gordon L. Hug and Prashant V. Kamat. (1995). Photochemistry on Surfaces. Intermolecular Energy and Electron Transfer Processes between excited Ru(bpy)32+ and H-Aggregates of Cresyl Violet on SiO2 and SnO2 Colloids. J. Phys. Chem. 99, 16768­16775. Retrieved July 23, 2012. Print.

[21] Tinoco, Ignacio Jr. (1960). Hypochromism in Polynucleotides. J. Phys. Chem. 82, 4785­ 4790. Retrieved July 23, 2012. Print.

[22] “Fluorescence quantum yield of cresyl violet in methanol and water as a function of concentration.” Stefan J. Isak and Edward M. Eyring. The Journal of Physical Chemistry 1992 96 (4), 1738-1742.

[23] Hardinger, Steve. "Chemistry Tutorial: Aromaticity." Organic Chemistry Tutorials. University of California, Los Angeles, n.d. Web. 18 June 2012. < http://www.chem.ucla.edu/harding/tutorials/aromaticity.pdf>.

[24] Guha, Rajarshi. (2001). Force Fields in Molecular Mechanics. Retrieved March 16, 2013, from http://rguha.net/writing/pub/ff.pdf.

[25] Molecular Modeling and Simulations. (2013). Chemical Computing Group. Retrieved March 16, 2013, from http://www.chemcomp.com/MOE- Molecular_Modeling_and _Simulations.htm.

[26] Healy, Eamonn F. (2007). Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy. J. Chem. Educ., 84(8), 1304. Retrieved August 15, 2012, from DOI: 10.1021/ed084p1304

[27] Dachineni, Rakesh. (2012). Interaction of Cresyl Violet with Nucleic Acids. Fairleigh Dickinson University. Print. Retrieved June 5, 2012.

[28] Williams, Alicia K., Sofia Cheliout Dasilva, Ankit Bhatta, Baibhav Rawal, Melinda Liu, and Ekaterina A. Korobkova. (2011). Determination of the drug-DNA binding modes using fluorescence-based assays. Analytical Biochemistry SciVerse ScienceDirect. Print. Retrieved September 25, 2012.

[29] Nazarenko, I., Pires, R., Lowe, B., Obaidy, M., and Rashtchian, A. (2002) Effect of Primary and Secondary Structure of oligodeoxyribonucleotides on the fluorescent properties of Conjugated dyes. Nuc. Acids. Res. 30:2089-2195.

[30] M.J. Waring, in: G.C.K. Roberts (Ed.), Drug Action at the Molecular Level,Macmillan, London, 1977, pp. 167-189.