Biomolecular engineering to make epigenetic knowledge useful
We are working to develop new methods for detecting gene-specific DNA methylation that are amenable to clinical use.
Non-coding, epigenetic modifications to DNA, in addition to the underlying ATCG code, have been shown to regulate gene transcription and play an important role in diseases such as cancer. Recent studies indicate that one such modification in promoter regions, DNA methylation, has value as a predictive biomarker. Current clinical practice relies on chemical conversion via bisulfite treatment to distinguish between methylated and unmethylated DNA; such treatment can suffer from inaccurate results due to incomplete conversion and DNA degradation. Previously, we developed a biochip-based method that can be used to detect DNA methylation without requiring chemical conversion in order to make this information available to doctors accurately and at low cost. To improve sensitivity and reduce the number of cells required for analysis, we are engineering methyl CpG binding domain (MBD) proteins to epigenotype fixed cells from biopsied tissue. Through this work, we seek to enable widespread epigenotyping in order to improve cancer diagnostics and patient outcomes and to speed the process of biomarker discovery and validation.
A methyl binding domain in complex with DNA bearing a symmetrically methylated CpG site. Methylation of cytosines in this sequence context in gene promoters leads to reduced expression of certain proteins in many cancers.
Biomolecular engineering to understand the role of redox biology in human disease
Reactive oxygen species are important in cell homeostasis as well as the development of human diseases. However, studies in redox biology have lacked a systematic approach due to deficiencies in the current-state-of-the-art tools for measuring and generating reactive species. It is important to be able to distinguish between particular chemical species, rather than lumping all reactive oxygen species together, and to measure intracellular concentrations with spatial and temporal resolution. We are using our skills in genetic engineering, protein engineering, and computational modeling to develop new tools to measure and generate reactive species with more precise chemical specificity and greater spatiotemporal resolution. Simultaneously, we are devising approaches for rigorous interpretation of signal readouts from currently available tools. We are applying our knowledge of these tools to achieve a more accurate understanding of ROS levels in diseases, which will aid in the development of effective therapeutics.
Reversible, genetically encoded sensors of hydrogen peroxide allow visualization and quantification of this oxidant in time and space.
Biomolecular engineering for affordable medical diagnostics
Immunoassay-based rapid diagnostic tests (RDTs) currently represent the leading means for point-of-care diagnosis of infectious diseases in developing contexts. These assays typically employ surface-immobilized IgG antibodies as affinity agents for the capture of disease-relevant biomarkers from patient samples. However, antibodies are prone to thermal denaturation and nonspecific binding events that can render test results unreliable, and their development is costly and time-intensive, which can increase costs to the end user. These characteristics limit the potential utility of antibody-based diagnostics in resource-constrained contexts, where cold chain storage may not be feasible and financial resources are limited. In order to better address these technological gaps, we are developing robust, minimalist, immuno-orthogonal affinity agents based on thermostable protein scaffolds, using yeast surface display and flow cytometric analysis. By incorporating these robust affinity agents into low-cost, multiplexed assay formats, we aim to enable the sensitive, specific, and timely diagnosis of high-priority infectious diseases at the point-of-care.
Polymer reaction engineering for affordable medical diagnostics
In addition to developing affinity agents better-suited for application in challenging environments, our group is seeking to further improve upon the RDT format by developing a new method of signal amplification for the transduction of positive binding events. Traditional colorimetric signal amplification methods such as enzymatic dye development or nanoparticle concentration are slow, they tend to produce low-contrast results, and they can produce false negative or false positive results if interpreted outside of the ideal time window for test development. In order to improve upon these techniques, our group has developed a novel means of signal read-out, termed photopolymerization-based amplification (PBA). This method, which uses a macrophotoinitiator conjugated to an affinity agent in the place of the dye-cleaving enzyme or gold nanoparticle, gives rise to a polymer film, visible to the unaided eye, upon exposure of a positive test strip to green light. PBA has been shown to yield a high-contrast, rapid, sensitive, and time-decoupled signal read-out, which could further enhance the suitability of RDTs for communities that could benefit. Our design choices made in incorporating PBA into the RDT format are informed by an awareness of the needs of the end-user, as well as a nuanced understanding of the reaction chemistry, developed over the course of studies exploring the capabilities of PBA on numerous surfaces and at varying reaction conditions.
Polymerization-based amplification transduces molecular recognition events into micron-scale hydrogel films that are visible by eye. These reactions proceed in air within thirty-five to one hundred seconds.