New materials for biological applications prepared by olefin metathesis reactions

With the advent of well-defined ruthenium olefin metathesis catalysts that are highly active and stable to a variety of functional groups, the synthesis of complex organic molecules and polymers is now possible; this is reviewed in Chapter 1. The majority of the rest of this thesis describes the application of these catalysts towards the synthesis of novel polymers that may be useful in biological applications and investigations into their efficacy.

A method was developed to produce polyethers by metathesis, and this is described in Chapters 2 and 3. An unsaturated 12-crown-4 analog was made by template- directed ring-closing metathesis (RCM) and utilized as a monomer for the synthesis of unsaturated polyethers by ring-opening metathesis polymerization (ROMP). The yields were high and a range of molecular weights was accessible. In a similar manner, substituted polyethers with various backbones were synthesized: polymers with benzo groups along the backbone and various concentrations of amino acids were prepared. The results from in vitro toxicity tests of the unsubstituted polyethers are considered.

The conditions necessary to synthesize polynorbornenes with pendent bioactive peptides were explored as illustrated in Chapter 4. First, the polymerization of various norbornenyl monomers substituted with glycine, alanine or penta(ethylene glycol) is described. Then, the syntheses of polymers substituted with peptides GRGD and SRN, components of a cell binding domain of fibronectin, using newly developed ruthenium initiators are discussed.

In Chapter 5, the syntheses of homopolymers and a copolymer containing GRGDS and PHSRN, the more active forms of the peptides, are described. The ability of the polymers to inhibit human dermal fibroblast cell adhesion to fibronectin was assayed using an in vitro competitive inhibition assay, and the results are discussed. It was discovered that the copoymer substituted with both GRGDS and PHSR peptides was more active than both the GRGDS-containing homopolymer and the GRGDS free peptide.

Historically, one of the drawbacks to using metathesis is the removal of the residual ruthenium at the completion of the reaction. Chapter 6 describes a method where the water soluble tris(hydroxymethyl)phosphine is utilized to facilitate the removal of residual ruthenium from RCM reaction products.

Label-free detection of single biological molecules using microtoroid optical resonators

Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the `reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

The biological sense of smell: olfactory search behavior and a metabolic view for olfactory perception

Part I of the thesis describes the olfactory searching and scanning behaviors of rats in a wind tunnel, and a detailed movement analysis of terrestrial arthropod olfactory scanning behavior. Olfactory scanning behaviors in rats may be a behavioral correlate to hippocampal place cell activity.

Part II focuses on the organization of olfactory perception, what it suggests about a natural order for chemicals in the environment, and what this in tum suggests about the organization of the olfactory system. A model of odor quality space (analogous to the "color wheel") is presented. This model defines relationships between odor qualities perceived by human subjects based on a quantitative similarity measure. Compounds containing Carbon, Nitrogen, or Sulfur elicit odors that are contiguous in this odor representation, which thus allows one to predict the broad class of odor qualities a compound is likely to elicit. Based on these findings, a natural organization for olfactory stimuli is hypothesized: the order provided by the metabolic process. This hypothesis is tested by comparing compounds that are structurally similar, perceptually similar, and metabolically similar in a psychophysical cross-adaptation paradigm. Metabolically similar compounds consistently evoked shifts in odor quality and intensity under cross-adaptation, while compounds that were structurally similar or perceptually similar did not. This suggests that the olfactory system may process metabolically similar compounds using the same neural pathways, and that metabolic similarity may be the fundamental metric about which olfactory processing is organized. In other words, the olfactory system may be organized around a biological basis.

The idea of a biological basis for olfactory perception represents a shift in how olfaction is understood. The biological view has predictive power while the current chemical view does not, and the biological view provides explanations for some of the most basic questions in olfaction, that are unanswered in the chemical view. Existing data do not disprove a biological view, and are consistent with basic hypotheses that arise from this viewpoint.

Improving the biological activity of pyrrole-imidazole polyamides

DNA is nature’s blueprint, holding within it the genetic code that defines the structure and function of an organism. A complex network of DNA-binding proteins called transcription factors can largely control the flow of information from DNA, so modulating the function of transcription factors is a promising approach for treating many diseases. Pyrrole-imidazole (Py-Im) polyamides are a class of DNA-binding oligomers, which can be synthetically programmed to bind a target sequence of DNA. Due to their unique shape complementarity and a series of favorable hydrogen bonding interactions that occur upon DNA-binding, Py-Im polyamides can bind to the minor groove of DNA with affinities comparable to transcription factors. Previous studies have demonstrated that these cell-permeable small molecules can enter cell nuclei and disrupt the transcription factor-DNA interface, thereby repressing transcription. As the use of Py-Im polyamides has significant potential as a type of modular therapeutic platform, the need for polyamides with extremely favorable biological properties and high potency will be essential. Described herein, a variety of studies have been performed aimed at improving the biological activity of Py-Im polyamides. To improve the biological potency and cellular uptake of these compounds, we have developed a next-generation class of polyamides bearing aryl-turn moieties, a simple structural modification that allows significant improvements in cellular uptake. This strategy was also applied to a panel of high-affinity cyclic Py-Im polyamides, again demonstrating the remarkable effect minor structural changes can have on biological activity. The solubility properties of Py-Im polyamides and use of formulating reagents with their treatment have also been examined. Finally, we describe the study of Py-Im polyamides as a potential artificial transcription factor.

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.

Tissue engineering active biological machines : bio-inspired design, directed self-assembly, and characterization of muscular pumps simulating the embryonic heart

Biological machines are active devices that are comprised of cells and other biological components. These functional devices are best suited for physiological environments that support cellular function and survival. Biological machines have the potential to revolutionize the engineering of biomedical devices intended for implantation, where the human body can provide the required physiological environment. For engineering such cell-based machines, bio-inspired design can serve as a guiding platform as it provides functionally proven designs that are attainable by living cells. In the present work, a systematic approach was used to tissue engineer one such machine by exclusively using biological building blocks and by employing a bio-inspired design. Valveless impedance pumps were constructed based on the working principles of the embryonic vertebrate heart and by using cells and tissue derived from rats. The function of these tissue-engineered muscular pumps was characterized by exploring their spatiotemporal and flow behavior in order to better understand the capabilities and limitations of cells when used as the engines of biological machines.

Synthesis and biological activity of anticoagulant heparan sulfate glycopolymers

Heparin has been used as an anticoagulant drug for more than 70 years. The global distribution of contaminated heparin in 2007, which resulted in adverse clinical effects and over 100 deaths, emphasizes the necessity for safer alternatives to animal-sourced heparin. The structural complexity and heterogeneity of animal-sourced heparin not only impedes safe access to these biologically active molecules, but also hinders investigations on the significance of structural constituents at a molecular level. Efficient methods for preparing new synthetic heparins with targeted biological activity are necessary not only to ensure clinical safety, but to optimize derivative design to minimize potential side effects. Low molecular weight heparins have become a reliable alternative to heparin, due to their predictable dosages, long half-lives, and reduced side effects. However, heparin oligosaccharide synthesis is a challenging endeavor due to the necessity for complex protecting group manipulation and stereoselective glycosidic linkage chemistry, which often result in lengthy synthetic routes and low yields. Recently, chemoenzymatic syntheses have produced targeted ultralow molecular weight heparins with high-efficiency, but continue to be restricted by the substrate specificities of enzymes.

To address the need for access to homogeneous, complex glycosaminoglycan structures, we have synthesized novel heparan sulfate glycopolymers with well-defined carbohydrate structures and tunable chain length through ring-opening metathesis polymerization chemistry. These polymers recapitulate the key features of anticoagulant heparan sulfate by displaying the sulfation pattern responsible for heparin’s anticoagulant activity. The use of polymerization chemistry greatly simplifies the synthesis of complex glycosaminoglycan structures, providing a facile method to generate homogeneous macromolecules with tunable biological and chemical properties. Through the use of in vitro chromogenic substrate assays and ex vivo clotting assays, we found that the HS glycopolymers exhibited anticoagulant activity in a sulfation pattern and length-dependent manner. Compared to heparin standards, our short polymers did not display any activity. However, our longer polymers were able to incorporate in vitro and ex vivo characteristics of both low-molecular-weight heparin derivatives and heparin, displaying hybrid anticoagulant properties. These studies emphasize the significance of sulfation pattern specificity in specific carbohydrate-protein interactions, and demonstrate the effectiveness of multivalent molecules in recapitulating the activity of natural polysaccharides.

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

Transition metal clusters supported by multinucleating ligand frameworks as models of biological active sites

This dissertation describes efforts to model biological active sites with small molecule clusters. The approach used took advantage of a multinucleating ligand to control the structure and nuclearity of the product complexes, allowing the study of many different homo- and heterometallic clusters. Chapter 2 describes the synthesis of the multinucleating hexapyridyl trialkoxy ligand used throughout this thesis and the synthesis of trinuclear first row transition metal complexes supported by this framework, with an emphasis on tricopper systems as models of biological multicopper oxidases. The magnetic susceptibility of these complexes were studied, and a linear relation was found between the Cu-O(alkoxide)-Cu angles and the antiferromagnetic coupling between copper centers. The triiron(II) and trizinc(II) complexes of the ligand were also isolated and structurally characterized.

Chapter 3 describes the synthesis of a series of heterometallic tetranuclear manganese dioxido complexes with various incorporated apical redox-inactive metal cations (M = Na⁺, Ca²⁺, Sr²⁺, Zn²⁺, Y³⁺). Chapter 4 presents the synthesis of heterometallic trimanganese(IV) tetraoxido complexes structurally related to the CaMn₃ subsite of the oxygen-evolving complex (OEC) of Photosystem II. The reduction potentials of these complexes were studied, and it was found that each isostructural series displays a linear correlation between the reduction potentials and the Lewis acidities of the incorporated redox-inactive metals. The slopes of the plotted lines for both the dioxido and tetraoxido clusters are the same, suggesting a more general relationship between the electrochemical potentials of heterometallic manganese oxido clusters and their “spectator” cations. Additionally, these studies suggest that Ca²⁺ plays a role in modulating the redox potential of the OEC for water oxidation.

Chapter 5 presents studies of the effects of the redox-inactive metals on the reactivities of the heterometallic manganese complexes discussed in Chapters 3 and 4. Oxygen atom transfer from the clusters to phosphines is studied; although the reactivity is kinetically controlled in the tetraoxido clusters, the dioxido clusters with more Lewis acidic metal ions (Y³⁺ vs. Ca²⁺) appear to be more reactive. Investigations of hydrogen atom transfer and electron transfer rates are also discussed.

Appendix A describes the synthesis, and metallation reactions of a new dinucleating bis(N-heterocyclic carbene)ligand framework. Dicopper(I) and dicobalt(II) complexes of this ligand were prepared and structurally characterized. A dinickel(I) dichloride complex was synthesized, reduced, and found to activate carbon dioxide. Appendix B describes preliminary efforts to desymmetrize the manganese oxido clusters via functionalization of the basal multinucleating ligand used in the preceding sections of this dissertation. Finally, Appendix C presents some partially characterized side products and unexpected structures that were isolated throughout the course of these studies.

Design, synthesis, and biological activity of rhodium metalloinsertors

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.

Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.

In addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.

To explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.

Finally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both Δ and Λ enantiomers of [Rh(chrysi)(phen)(DPE)]²⁺ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.

On the Role of Delays in Biological Systems : Analysis and Design

This work quantifies the nature of delays in genetic regulatory networks and their effect on system dynamics. It is known that a time lag can emerge from a sequence of biochemical reactions. Applying this modeling framework to the protein production processes, delay distributions are derived in a stochastic (probability density function) and deterministic setting (impulse function), whilst being shown to be equivalent under different assumptions. The dependence of the distribution properties on rate constants, gene length, and time-varying temperatures is investigated. Overall, the distribution of the delay in the context of protein production processes is shown to be highly dependent on the size of the genes and mRNA strands as well as the reaction rates. Results suggest longer genes have delay distributions with a smaller relative variance, and hence, less uncertainty in the completion times, however, they lead to larger delays. On the other hand large uncertainties may actually play a positive role, as broader distributions can lead to larger stability regions when this formalization of the protein production delays is incorporated into a feedback system.

Furthermore, evidence suggests that delays may play a role as an explicit design into existing controlling mechanisms. Accordingly, the reccurring dual-feedback motif is also investigated with delays incorporated into the feedback channels. The dual-delayed feedback is shown to have stabilizing effects through a control theoretic approach. Lastly, a distributed delay based controller design method is proposed as a potential design tool. In a preliminary study, the dual-delayed feedback system re-emerges as an effective controller design.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.