McGown Research Group
Research
Current research interests in the McGown group include G-quadruplex formation in genomic DNA and its role in normal
and pathological gene regulation, design and applications of reversible biogel materials for bio- and nano-encapsulation,
affinity capillary electrophoresis and affinity MALDI mass spectrometry platforms for directed proteomic discovery of
biomarkers, analytical applications of aptamers and genomic inspired oligonucleotides, gel electrochromatography for
bioseparations, DNA and genomic analysis, and fundamental studies of molecular aggregattion and self-assembled media.
Areas of applications include medicine, biotechnology, nanotechnology, forensics, environmental analysis, and chemical
and biological detection.
A recurring theme in these projects is the involvement of G-quadruplex DNA. In some cases, we are investigating guanine-rich oligonucleotides that fold into intramolecular G-quadruplex structures. Examples include certain aptamers such as the thrombin-binding
aptamer, and genome-inspired oligonucleotides derived from sequences found for example in promoter regions of many genes and in
telomeric DNA. We are particularly interested in protein capture by these G-quadruplex strucutres for affinity analysis,
biomarker discovery and fundamental biological studies. In other cases, we are investigating applications of guanosine gels (G-gels).
These are self-assembled networks of hydrogen-bonded guanine tetrads formed by guanosine nucleosides and nucleotides. G-gels combine
desirable properties, such as reversibility, tunability, aqueous solubility, and biocompatibility, with the unique ability to non-covalently
and reversibly introduce functionality directly into the G-tetrad network of the gel via hydrogen bonding. Their degree of organization
and viscosity are dependent upon monomer concentration, temperature, pH and cation content, providing a variety of parameters that can be u
sed to control their formation/disassembly and to reversibly modulate their properties. We are exploring applications of guanosine gels in
chemical and biological separations, bioencapsulation and formation of nanodevices.
G-Quadruplex DNA and Insulin Proteins in Type 1 Diabetes
Genome-inspired Affinity Platforms for Biomarker Discovery
A Genome-Inspired DNA Ligand for Affinity Capture of Insulin Proteins
Genomic G-quadruplex DNA in Cancer
Design and Evaluation of Thermoassociative Guanosine Gels
Encapsulation of Living Cells in G-gels
G-Gel Phases for Biological and Chemical Separations
Affinity binding reagents have played a crucial role in the translation of proteomic discoveries to clinical
diagnostics due to their ability to isolate target proteins from complex protein mixtures. Antibodies have been
unrivaled as affinity reagents for proteins due to their strong and selective binding; however, drawbacks
associated with their production, stability and manipulation have prompted researchers to seek alternatives.
Foremost among alternatives are aptamers, which offer affinity on par with that of monoclonal antibodies, but
with important advantages: first, once an aptamer to a target protein has been identified, it can be synthesized,
chemically modified and manipulated with ease; second, aptamers are chemically stable and can be reversibly folded
and unfolded for capture and release of the target protein, allowing aptamer-modified surfaces to be reused indefinitely.
Our group is exploring aptamer-modified surfaces for affinity protein capture and detection in capillary electrophoresis
and in Matrix-Assisted Laser Desorption-Ionization Mass Spectroscopy (MALDI-MS). Using our new approaches we have demonstrated
capture and detection of thrombin in human serum, of insulin proteins in nuclear extracts of cell lysates and of immunoglobulin
E in human serum.
(Collaboration with Professor Lee Ligon in the Department of Biology)
The long term goal of this research program is to elucidate the molecular cell biology contributing to the development of Type 1 Diabetes (IDDM). Studies of nuclear localization and intranuclear function of polypeptide hormones and growth factors in recent years establish the basis for re-examining long-held beliefs about nuclear translocation and intranuclear function of polypeptides, including insulin and insulin-like growth factor (IGF) proteins. Based on these recent developments and our own preliminary results demonstrating the presence of insulin in the nuclei of human fetal thymus cells and its association there with DNA in the insulin-linked polymorphic region (ILPR) of the insulin gene promoter region, we have hypothesized an insulin pathway that incorporates unique nuclear entrance of insulin and insulin as a direct mediator of its own expression through binding with G-quadruplex structures in the ILPR. We are testing this hypothesis in systematic in vitro and in vivo studies using analytical chemistry and molecular cell biology. Elucidation of the molecular basis of IDDM will lead to new strategies for its prediction, treatment and prevention. The results will lead to new, fundamental insights into the role of genomic architecture such as the G-quadruplex in regulation of gene expression and genetic disease at the molecular level. It may also increase our understanding of other autoimmune diseases.
This project combines new tools for protein capture with a “directed proteomic” strategy for protein profiling that targets proteins that bind to G-quadruplex DNA formed by sequences derived from guanine rich regions of the human genome. We have established two approaches to protein capture. In the first, the individual G-quadruplex DNA oligonucleotides are immobilized at inner surfaces of fused silica capillaries for protein capture for use in affinity capillary electrophoresis. In the second, arrays of G-quadruplex-forming oligonucleotides are immobilized at MALDI probe surfaces for affinity MALDI mass spectrometry. We have established that the immobilized oligonucleotides assume G-quadruplex conformations at the fused silica surfaces, and we have demonstrated the effectiveness of both approaches for affinity capture of G-quadruplex binding proteins. The novel use of specific DNA structural motifs drawn from genomic DNA as a target for protein binding could be extended to other DNA structures as well. The result will be discovery of new binding ligands (as exemplified by our recent discovery of an insulin-binding ligand) that could be used not only in affinity analysis but also, arising from the use of genome-inspired sequences, for biomarker discovery and exploration of binding interactions and structural genomics in living cells.
We have identified a genome-inspired DNA ligand that exhibits selective binding towards insulin and related insulin-like proteins. The immobilized ligand was able to capture insulin from complex matrices such as nuclear extracts and cell lysates with high selectivity, in both affinity MALDI-mass spectrometry and affinity capillary electrophoresis. The availability of a DNA binding ligand to human insulin offers an alternative to antibodies for in vitro or in vivo detection and sensing of insulin as well as its isolation and purification from biological samples. Although the DNA binding ligand to insulin was not derived combinatorially and is not therefore an “aptamer”, it offers the same advantages such as ease of production and manipulation, stability, relatively small size, and re-usability. In addition to insulin capture and detection, we will explore the use of a labeled insulin analog for detection of G-quadruplex formation by genomic DNA in the nuclei of living cells.
Important advances in detection and diagnosis of cancer have resulted from the use of proteomic approaches to identify protein biomarkers through empirical, comprehensive comparisons of cellular proteomes. We are taking an alternative approach that is based on the biological hypothesis that G-quadruplex DNA structures in guanine-rich regions of human chromosomes are linked to regulation of nuclear processes that go awry in cancer and that this pathology will be reported by changes in the cellular proteome. Specifically, we are seeking to identify nuclear proteins that bind to genomic G-quadruplex DNA formed by G-rich sequences from promoter regions of human oncogenes. Rapid screening is achieved using Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry to detect captured proteins on arrays of immobilized G-quadruplex DNA. Proteins of interest can then sequenced and identified. This research has the potential not only to discover new protein biomarkers of cellular transformations related to the onset and development of cancer, but also to provide new insights into the role of genomic architecture in these transformations. The results of this project will potentially lead to new approaches to early detection of cancer and new strategies for its prevention and treatment.
It is well known that guanosine nucleotides and their derivatives form gels in aqueous solution, as a result of hydrogen
bonding between a guanine base and its nearest neighbors. Typically these gels are thermothinning; they exhibit a strict
decrease in viscosity with an increase in temperature. We have discovered thermoassociative guanosine gels (G-gels) that
exhibit an increase in viscosity with an increase in temperature. These G-gels are created from mixtures of guanosine
compounds and differ from previously reported G-gels as a result of their thermoassociative nature. Using spectroscopic
techniques, we can monitor gel aggregation, melting behavior and reversibility as a function of temperature. This unique
inverse thermal dependence makes these types of gels ideal for encapsulation of heat sensitive components such as living
cells, enzymes and other biological components, since they could be added to the solution at low temperature for homogeneous
distribution and then raised to room or body temperature for gelation into, for example, drug delivery devices, artificial
cells, tissues and organs, and media for environmental bioremediation. Similarly, nanocomponents could be dispersed and
stored in the solutions at low temperatures and then immobilized in the gels for use at higher temperatures.
(in collaboration with Professor Jan Stegemann in the Bioengineering Department)
Much progress has been made in recent years in the design and construction of materials for cell encapsulation for medical
applications such as delivery of therapeutic agents and the construction of artificial tissues and organs. Advances in the
design and construction of cell encapsulation media, the vast majority of which are based on gels formed by natural or synthetic
polymers, have yielded promising results. Nevertheless, materials have yet to emerge that fully satisfy the requirements for in
vivo applications: mechanical strength and durability, long-term stability, biocompatibility, cell viability, low immunogenicity,
availability in high purity, standardized forms, and economic feasibility. We are investigating gels formed by reversible self-association
of guanosine compounds, referred to as G-gels, for cell encapsulation. We recently discovered that mixtures of guanosine compounds in
certain proportions form G-gels that are thermoassociative, in other words, they are solutions at low temperature and become gel-like
upon heating. These thermoassociative G-gels offer important potential advantages for cell encapsulation, including ease of formation
by simple, relatively low cost, small molecular compounds, reversible gelation, ease of cell incorporation under gentle conditions,
potentially low toxicity and immunogenicity, and stability at physiological temperatures in the range of neutral to low pH. As with
polymeric hydrogels, structure and strength may be augmented by incorporation of additional components such as nanoparticles.
In this project we are investigating G-gels as mobile phases for Capillary Gel Electrokinetic Chromatography (CGEKC).
Applications of current interest include separations of chiral compounds, DNA oligonucleotides and proteins. Most recently,
we applied G-gels to separation of four DNA 76-mers that are part of a highly polymorphic short tandem repeat commonly used
in comprehensive DNA testing in forensic investigations. The four sequences differ from each other by only a few G-A substitutions.
The number and location of these substitutions varies for each sequence, providing a mechanism for exploring G-gel recognition towards
adenosine or guanosine nucleotides in each strand. This is of particular interest given the possibility that guanosines in the DNA
might interact with the GMP in the gel phase to form transient G-tetrads as the oligonucleotides migrate through the G-gel. The results
demonstrate the effectiveness of G-gels for separation of oligonucleotides of identical length based solely on minor difference in
sequence. We are now investigating G-gels in capillary and slab electrophoresis formats for separations of longer DNA and for protein
separations that have proven difficult for existing techniques.