The Fordyce Lab is focused on developing new instrumentation and assays for making systems-scale biophysical measurements of molecular interactions. These tools have many diverse applications, but share a common approach: using techniques and ideas from physics and engineering to improve our quantitative understanding of fundamental biological processes. Experiments in the lab build upon two technology platforms we have developed:
MITOMI (Mechanically-Induced Trapping of Molecular Interactions) relies on spatial multiplexing to keep track of analytes throughout an experiment. We are currently using these devices to better understand how transcription factors find and bind their genomic targets to regulate gene expression, as well as to understand how enzymes achieve their extraordinary catalytic efficiency and substrate specificity.
MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) rely on spectral multiplexing to track analytes throughout an experiment. In these assays, we can create microspheres containing > 1,000 unique ratios of lanthanide nanophosphors that can be uniquely identified via imaging alone. We are currently developing new assays that use these microspheres to understand how signaling proteins recognize their peptide substrates and to improve our ability to extract information from single cells.
Probing how transcription factors find and bind their genomic targets
Gene expression is governed by binding of transcription factor proteins to regulatory sequences in the genome, where they either recruit or block transcriptional machinery to activate or repress expression. Despite many years of research, we remain largely unable to predict where transcription factors bind in vivo, and current models cannot explain how closely related transcription factors often regulate very distinct sets of target genes. We are combining the MITOMI platform with tools from chemical biology to precisely dissect the biophysical mechanisms by which transcription factors recognize DNA and improve our ability to predict these essential interactions.
Revealing how kinetics of transcription factor binding affect occupancy and gene expression
Recent evidence suggests that non-equilibrium binding dynamics may govern transcription factor activity at regulatory sites in the genome. Despite this fact, nearly all investigations of transcription factor binding in vitro measure only binding at equilibrium, without observing kinetics. We are developing a high-throughput microfluidic assay that can measure the residency time of molecular interactions. We expect this assay will be compatible with both extracted DNA and chromatin, allowing for investigations of the native epigenetic contexts that shape gene expression.
Understanding how enzymes achieve their extraordinary catalytic efficiency and specificity
Enzymes are the most efficient catalysts known, enhancing specific reaction rates by up to 17 orders of magnitude. However, we do not fully understand how they achieve this tremendous catalytic efficiency, which limits our ability to understand their biological function and design new enzymes with industrially or medically important applications. In close collaboration with the Herschlag lab, we are using the MITOMI platform to enable high-throughput investigation of how individual residues throughout an enzyme contribute to catalysis. This new platform allows detailed biochemical characterization of thousands of enzymes in parallel, enabling structure/function studies of enzymes at unprecedented scale.
Automating printing and image acquisition and analysis
Uncovering the biophysical mechanisms that drive transcription factor binding requires the ability to efficiently and reproducibly collect and compare data from many proteins. To streamline this process, we have been automating all asepcts of the experiments, from rebuilding old microarray printers to developing new software for valve automation and image processing.
A high-throughput assay for profiling protein-peptide interactions
While advances in deep sequencing have dramatically improved our ability to profile how DNA and RNA binding proteins interact with their substrates, we lack similar tools for quantitative, high-throughput investigation of protein-peptide interactions. We have recently demonstrated that peptides can be synthesized directly on our encoded MRBLEs, laying the foundation for novel high-throughput on-bead assays to probe how proteins recognize their peptide substrates. In collaboration with the Cyert Lab, we are using this platform to understand how calcineurin, a human phosphatase essential for the immune response and the target of several immunosuppresents, binds its targets.
A cheap and scalable way to synthesize custom peptide libraries on encoded beads
The ability to flexible construct specific peptide libraries on spectrally encoded beads would provide a powerful proteomic tool. Using a commercial video projector ($300, Amazon.com), we are developing a new system that uses patterned light to synthesize specific peptide sequences onto beads spatially arrayed within a microfluidic device.
Using encoded beads to link genotype and phenotype for many single cells in parallel
Recent developments in single-cell analysis have enabled the probing of phenotypes, transcriptomes, and genomes for individual cells, revealing previously hidden heterogeneity within cell populations of high significance for human health and disease. The next great challenge is to understand both the cause and effect of this heterogeneity by linking changes in genetic information with their functional effects. We are developing a new technologies that uses oligonucleotide arrays coupled to encoding beads to link different types of measurements for the same cell.
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