Jena Jacobs, Jessica Daniel*, Aviva Bulow, Susan Jett*, Derek Clark, Jonathan Richards*, Laura Roon*, Kathryn Norquest*, Stephen Schaffner*, Ryan Masterson*, Ilia Mazin, Nazar Dubchak, Ryan Warren, Tonya Santaus, Kyra Brandt, Elina Baravik*, Josh Sowick, Becky Addison, Jody Stephens*, Yerelsy Reyna*, Jeremy O’Brien, Travis Ingraham, Matthew Stoddard, Morgan Miller, Amanda Faux, Mason Preusser, Sarai Graves, Tiffany Ashbaugh, Michael McCoy, Ebony Miller
(* denotes a researcher with a publication from the lab)
Dr. Andrew J. Bonham
Associate Professor of Chemistry
Dr. Bonham’s Curriculum Vitae
Dr. Bonham’s work focuses on understanding and investigating Transcription Factors, essential human proteins that regulate the bodies growth and response to disease. These transcription factors are essential components of gene regulation, and there is great interest in probing their presence and activity in both academic analysis and clinical diagnostics. Current methods to address these questions are often time-intensive or require specialized reagents, such as antibodies. At Metropolitan State University of Denver, Dr. Bonham is leading an innovative undergraduate research program focused on engineering new tools for sensitive and quick detection of TF:DNA interactions.
Lab Member 2014-
Current Project: Selecting aptamers for the detection of the cancer biomarker ENOX2
Former Project: Expanding electrochemical DNA biosensors to detect ricin
Ricin toxin chain A (RTA), a byproduct of the production of castor oil from castor bean plants, is a hazardous toxin that inhibits the cellular production of proteins once it enters the body. This toxin, whether ingested, inhaled, or injected, can be lethal, and treatment is difficult as there are currently no known antidotes for ricin. Detection of RTA prior to exposure is thus important, and it is necessary to expand the methods that can be used for this detection, as current methods are not time-sensitive. In this project, we have designed and tested an electrochemical biosensor that is capable of– and sensitive enough– to detect small, bio-medically relevant concentrations of RTA. This biosensor was designed based on an existing oligonucleotide aptamers that have been previously shown to bind to the hydrolase protein in ricin toxin. One of these aptamers was then used as the basis of a rationally designed DNA oligonucleotide biosensor scaffold that allows the coupling of RTA binding to a conformational change in the oligonucleotide. Ultimately, this biosensor design allows voltammetric interrogation to detect RTA concentration in complex media (such as coffee, blood, and river water). Additionally, this biosensor is convenient and collects real-time data, offering beneficial applications to the monitoring processes of areas involved in castor oil production. Furthermore, it may possess diagnostic potential in assessing ricin exposure. Electrochemical DNA biosensors have the potential to be used in numerous different situations, and this project shows a strategy for how they may be expanded to the detection and quantification of hazardous toxins.
Cardiomyopathies, diseases of the heart, are one of the major causes of death in the United States, and thus there is great interest in preventing and treating these complications. However, due to the nature of limited availability of donors, many techniques and solutions are inadequate to meet the needs of the field. In particular, heart tissue transplantation, culturing human tissues/tissue-derived cells and tissue engineering, and finding a suitable extracellular environment that closely resembles the natural environment of cardiomyocytes in vivo are difficult to attain. As such, a great deal of work has gone into efforts to produce polymers which mimic the natural cell environment in properties such as binding sites, stiffness, reactivity, and hydration. In our research, we are investigating the development and characterization of conductive polymer scaffolds providing cardiac tissue support, which will ideally aid in culturing cardiomyocytes for academic, research, and medical use. The core of our model is the incorporation of conductive gold nanorods into reverse thermal gel polymers. Synthesized gold nanorods utilized in this procedure are made with a high aspect ratio, at high purity and with defined surface functionalization. These conducting scaffolds should properly accommodate cardiac cells in building functional cardiac tissue constructs and further improving cell retention, spreading, homogenous distribution of cardiac specific markers, cell-cell coupling and synchronized beating behavior at the tissue level. The nanoparticles have been synthesized, then covalently coupled into these scaffolds, resulting in order-of-magnitude increases in conductivity. The goal of this work aims to improve cardiac tissue engineering, so that it can be directed to ultimately repairing damaged heart muscle and improve overall cardiac function in cardiovascular diseases whether they have been acquired from past medical complications or developed through hereditary traits.
Celiac disease is an autoimmune disorder where ingesting dietary gluten causes an autoimmune response that leads to villous atrophy and other complications. Symptoms can have gastrointestinal or extraintestinal manifestations. The symptoms can include: diarrhea, abdominal pain, dental enamel defects, neurological issues, Dermatitis Herpetiformis and more. This disease affects an estimated 2 million Americans, 1.4 million of which are undiagnosed1. Rate of diagnosis is low because many affected individuals have “silent celiac disease” where the symptoms are not detectable, but villous atrophy is still occurring. Also, there is a broad spectrum of symptoms for the condition and is often misdiagnosed. The average time it takes to diagnosis individuals who are symptomatic in the US is 11 years2. In that time, their risk of developing other autoimmune disorders, neurological problems, fertility problems and other conditions increases. One key challenge of this condition is that the diagnosis has historically suffered from difficulties in performing non-invasive positive identification of affected patients. Recently, interacting amino acid sequences on the surface of human tissue transglutaminase and gliadin proteins from foodstuffs have been identified as the potential recognition sites of commonly developed auto-antibodies in Celiac disease. In this project, we are exploring the use of short synthetic peptides that mimic these key amino acid recognition sites in order to create rapid, non-invasive, electrochemical biosensors to aid in the early diagnosis of Celiac disease. These biosensors build on a literature of electrochemical biosensors that offer reagentless, reusable, and rapid testing directly in small quantities of blood (such as a finger lancet draw). Our goal is to design, synthesize, and validate such diagnostic biosensors.
Electrochemical biosensors based on the conformational dynamics of DNA aptamers have found success against a wide variety of proteins, toxins, antibodies, and heavy metals. However, the mechanistic underpinnings of the mechanism by which these surface-bound DNA molecules change conformation upon target binding, thus changing the dynamics of an appended redox-active tag and generating a measurable signal, is poorly understood. Our first target for investigation was a previously reported Ricin Chain A binding atamer biosensor. Here, we have investigated this biosensor using a variety of nucleic acid assays, including PCR-termination via basepair modification, fluorescence anisotropy, gel mobility shift assays, and FRET tagging to determine 3D orientation. These have allowed us to better characterize the basepair interactions involved in binding targets, as well as offer clues to the changing three dimensional folded structures of these biosensors. These results will help inform the field of biosensors and aptamers in general on strategies for future optimization.
Oligonucleotide-based biosensors have been demonstrated as effective tools for detecting heavy metals, small molecule drugs, and protein targets. Recent efforts to move these biosensors from the lab bench to practical use in industry and medicine rely on the ability to rapidly and effectively adapt them to detect new targets of interest. One compelling aspect of DNA and RNA-based biosensors is the great strides that have been made in computational prediction of the three-dimensional structures that they can assume. However, interpreting predicted structures and using that knowledge to design functional biosensors ab initio remains a challenging problem. We hypothesize that by representing a potential DNA biosensor as a node-weighted graph, we can simplify the challenge of automated structure interpretation and sorting. If correct, this would allow a researcher to provide a core sequence of interest (such as a DNA recognition element or artificially-selected aptamer) and have a software tool design the ideal DNA biosensor for the sensitive detection of that target. This process builds on ideas from our oligionucleotide fold scoring algorithm, Fealden. With this new implementation of Fealden, we are achieving significantly more flexibility, allowing us to analyze any structure type. Because of this we are able to traverse a search space of tens of thousands of possible sequences to determine a “best candidate” biosensor. Additionally, with this increased flexibility we hope to implement some very desirable features in our software, generally, different types of design constraints that a researcher can specify for their biosensor.
One of the many great challenges that medical diagnostics face is the need for sensitive, reliable, and rapid detection of molecules in very complex solutions such as blood or urine. DNA-based biosensors have shown great promise in terms of sensitivity and reliability for target detection, but the need for rapid testing has considerably slowed their use in practical applications within the medical world. In the research to be conducted, we explore the incorporation of DNA-based biosensors into a lateral flow assay format (similar to the common at-home pregnancy test for human chorionic gonadotropin in urine). To facilitate this, we are developing a gold nanoparticle decorated with a functional DNA probe that recognizes and binds to botulism neurotoxin variant A (BoNTA). This conjugate then wicks across a nitrocellulose membrane to specific capture points, allowing rapid visual assessment of the BoNTA contamination of a sample. In the future, we aim to demonstrate that this represents a generic platform for detection that could be used with any existing DNA aptamer-based biosensing technique and can be applied to many medical settings, including small clinics, without the need for technicians to operate the biosensor.