AI-guided materials discovery & automated chemical synthesis
We are entering a technological revolution in automated chemical synthesis. For many years, sequence-defined biomolecules such as peptides and oligonucleotides have been synthesized using automated techniques, but similar methods for synthetic molecules lags far behind. Precise control of chemical sequence in oligomeric and polymeric materials plays a direct role in bulk material properties such as charge transport and self-assembled structures. Traditionally, access to sequence-defined synthetic molecules has been limited to only a handful of skilled chemists performing time-consuming specialized syntheses. In our group, we are using high-throughput, automated synthesis techniques to access sequence-defined, synthetic oligomers. We built a custom, automated Molecule Maker instrument to perform these syntheses for a wide range of chemistries and applications in electronic materials, fluorescent dyes, and gels.
Flavin-binding fluorescent proteins (FbFPs) for anaerobic imaging
Recently, a new class of oxygen-independent fluorescent reporters was reported based on flavin-binding photosensors from Bacillus subtilis, Pseudomonas putida, and Arabidopsis thaliana. Flavin-binding fluorescent proteins (FbFPs) can function in the absence of oxygen, whereas the widely used green fluorescent protein (GFP) and related analogs strictly require oxygen for maturation of fluorescence. In our lab, we have applied the tools of directed evolution to isolate new and spectrally improved variants of FbFPs. Moving forward, we anticipate that spectrally enhanced FbFP variants will find pervasive use as reporter proteins for gene expression, subcellular localization and protein interactions in obligate and facultative anaerobes and in hypoxic niches of the human body (e.g. malignant tumors).
1. A. Mukherjee, K. B. Weyant, J. Walker, U. Agrawal, I. Cann, C. M. Schroeder, “Engineering and Characterization of New LOV-based Fluorescent Proteins from Chlamydomonas reinhardtii and Vaucheria frigida“, ACS Synthetic Biology, 4, 371 (2015).
2. A. Mukherjee, J. Walker, K. B. Weyant, C. M. Schroeder, “Characterization of Flavin-based Fluorescent Proteins: An Emerging Class of Powerful Fluorescent Reporters”, PLOS ONE, 8, e64753 (2013).
3. A. Mukherjee, K. B. Weyant, J. Walker, C. M. Schroeder, “Directed Evolution of Bright Mutants of a Flavin-Dependent Anaerobic Fluorescent Protein from Pseudomonas putida“, Journal of Biological Engineering, 6, 20, (2012).
4. A. Mukherjee and C. M. Schroeder, “Flavin-based Fluorescent Proteins: Emerging Paradigms in Biological Imaging”, Current Opinion in Biotechnology, 31, 16 (2015).
Fluorescent dendrimer nanoconjugates
Advanced imaging techniques in the biological and chemical sciences critically rely on bright and photostable probes. We developed a new class of fluorescent molecules, fluorescent dendrimer nanoconjugates (FDNs), based on the covalent attachment of multiple fluorescent dyes onto a single dendritic scaffold. This results in extremely bright, nanometer scale, single-molecule probes that we have used for both traditional fluorescence microscopy, as well as for the burgeoning field of super-resolution microscopy where brightness is directly related to achievable resolution. In addition, we have engineered the photophysical properties of these probes by attaching the “photo-protective” triplet-state quencher Trolox directly onto the scaffold, an anti-fade agent normally used in solution. Trolox-conjugated probes are extremely photostable with vastly decreased amounts of transient dark states compared with single fluorescent dyes, and by modulating the amount of conjugated Trolox, we can exceed enhancement in photostability provided by adding large concentrations of Trolox in solution.
1. D. T. Reilly, S. H. Kim, J. A. Katzenellenbogen, C. M. Schroeder, “Fluorescent Nanoconjugate Derivatives with Enhanced Photostability for Single Molecule Imaging”, Analytical Chemistry, 87, 11048 (2015).
2. Y. Kim, S. Kim, M. Tanyeri, J. A. Katzenellenbogen, C. M. Schroeder, Dendrimer Probes for Enhanced Photostability and Localization in Fluorescence Imaging, Biophysical Journal, 104, 1566 (2013).