Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Structure Prediction of G-Protein Coupled Receptors

G-protein coupled receptors (GPCRs) form a large family of proteins and are very important drug targets. They are membrane proteins, which makes computational prediction of their structure challenging. Homology modeling is further complicated by low sequence similarly of the GPCR superfamily.

In this dissertation, we analyze the conserved inter-helical contacts of recently solved crystal structures, and we develop a unified sequence-structural alignment of the GPCR superfamily. We use this method to align 817 human GPCRs, 399 of which are nonolfactory. This alignment can be used to generate high quality homology models for the 817 GPCRs.

To refine the provided GPCR homology models we developed the Trihelix sampling method. We use a multi-scale approach to simplify the problem by treating the transmembrane helices as rigid bodies. In contrast to Monte Carlo structure prediction methods, the Trihelix method does a complete local sampling using discretized coordinates for the transmembrane helices. We validate the method on existing structures and apply it to predict the structure of the lactate receptor, HCAR1. For this receptor, we also build extracellular loops by taking into account constraints from three disulfide bonds. Docking of lactate and 3,5-dihydroxybenzoic acid shows likely involvement of three Arg residues on different transmembrane helices in binding a single ligand molecule.

Protein structure prediction relies on accurate force fields. We next present an effort to improve the quality of charge assignment for large atomic models. In particular, we introduce the formalism of the polarizable charge equilibration scheme (PQEQ) and we describe its implementation in the molecular simulation package Lammps. PQEQ allows fast on the fly charge assignment even for reactive force fields.

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.

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.

Model systems for the study of drug hypersensitivity

I. It was not possible to produce anti-tetracycline antibody in laboratory animals by any of the methods tried. Tetracycline protein conjugates were prepared and characterized. It was shown that previous reports of the detection of anti-tetracycline antibody by in vitro-methods were in error. Tetracycline precipitates non-specifically with serum proteins. The anaphylactic reaction reported was the result of misinterpretation, since the observations were inconsistent with the known mechanism of anaphylaxis and the supposed antibody would not sensitize guinea pig skin. The hemagglutination reaction was not reproducible and was extremely sensitive to minute amounts of microbial contamination. Both free tetracyclines and the conjugates were found to be poor antigens.

II. Anti-aspiryl antibodies were produced in rabbits using 3 protein carriers. The method of inhibition of precipitation was used to determine the specificity of the antibody produced. ε-Aminocaproate was found to be the most effective inhibitor of the haptens tested, indicating that the combining hapten of the protein is ε-aspiryl-lysyl. Free aspirin and salicylates were poor inhibitors and did not combine with the antibody to a significant extent. The ortho group was found to participate in the binding to antibody. The average binding constants were measured.

Normal rabbit serum was acetylated by aspirin under in vitro conditions, which are similar to physiological conditions. The extent of acetylation was determined by immunochemical tests. The acetylated serum proteins were shown to be potent antigens in rabbits. It was also shown that aspiryl proteins were partially acetylated. The relation of these results to human aspirin intolerance is discussed.

III. Aspirin did not induce contact sensitivity in guinea pigs when they were immunized by techniques that induce sensitivity with other reactive compounds. The acetylation mechanism is not relevant to this type of hypersensitivity, since sensitivity is not produced by potent acetylating agents like acetyl chloride and acetic anhydride. Aspiryl chloride, a totally artificial system, is a good sensitizer. Its specificity was examined.

IV. Protein conjugates were prepared with p-aminosalicylic acid and various carriers using azo, carbodiimide and mixed anhydride coupling. These antigens were injected into rabbits and guinea pigs and no anti-hapten IgG or IgM response was obtained. Delayed hypersensitivity was produced in guinea pigs by immunization with the conjugates, and its specificity was determined. Guinea pigs were not sensitized by either injections or topical application of p-amino-salicylic acid or p-aminosalicylate.

Understanding Co-translational Protein Targeting and Lithium Dendrite Formation through Free Energy Simulations and Coarse-Grained Models

We describe the application of alchemical free energy methods and coarse-grained models to study two key problems: (i) co-translational protein targeting and insertion to direct membrane proteins to the endoplasmic reticulum for proper localization and folding, (ii) lithium dendrite formation during recharging of lithium metal batteries. We show that conformational changes in the signal recognition particle, a central component of the protein targeting machinery, confer additional specificity during the the recognition of signal sequences. We then develop a three-dimensional coarse-grained model to study the long-timescale dynamics of membrane protein integration at the translocon and a framework for the calculation of binding free energies between the ribosome and translocon. Finally, we develop a coarse-grained model to capture the dynamics of lithium deposition and dissolution at the electrode interface with time-dependent voltages to show that pulse plating and reverse pulse plating methods can mitigate dendrite growth.