Assistant Professor, Department of Chemistry
Fahie, M., Chisholm, C., & Chen, M. (2015). Resolved Single-Molecule Detection of Individual Species within a Mixture of anti-Biotin Antibodies Using an Engineered Monomeric Nanopore. ACS Nano, 9(2), 1089–1098. https://doi.org/10.1021/nn506606e
Detecting rare biomolecules in complex samples like blood, soil and food is like finding the proverbial needle in a haystack. Billions of dollars are spent every year trying to isolate and quantify pathogens, cancer biomarkers and contaminants with a reliable assay. Nanopores can detect these molecules, but existing nanopores are limited in their ability to distinguish among similar molecules, and are also expensive to manufacture. Developing low-cost tests that are robust in their ability to sense different targets will help meet the needs of medical, environmental and biodefense industries, thus opening up vast markets.
This study presents a next-generation advance in nanopore-based biosensing. It presents a flexible nanopore, derived from E. coli bacteria, whose dynamic interface generates a highly-specific analyte fingerprint. This nanopore is the first to be sensitive enough to distinguish between nearly identical protein isoforms; it can also detect molecules that are larger than the pore. It is also much simpler to engineer than other nanopores, and may be rapidly generated. The authors are already collaborating with Oxford Nanopore Technologies to explore the new class of nanopores as industry-ready detectors.
Read more about Min Chen.
Associate Professor, Department of Biochemistry and Molecular Biology
Joshi, K. K., Bergé, M., Radhakrishnan, S. K., Viollier, P. H., & Chien, P. (2015). An Adaptor Hierarchy Regulates Proteolysis during a Bacterial Cell Cycle. Cell, 163(2), 419–431. https://doi.org/10.1016/j.cell.2015.09.030
The alarming increase in the number of antibiotic-resistant bacterial infections has made the discovery of new antibiotics a critical human health priority. Why have drug resistant bacteria emerged so quickly? One major factor is that all current antibiotics work by killing bacteria completely. While it is true that dead bacteria cannot infect hosts, this approach also creates tremendous pressure for the bacteria to evolve resistance mechanisms. Give that the human microbiome consists of billions of bacteria that are cordially living with their host, it is clear that the problem is not the simple presence of bacteria, but rather certain bacteria in a virulent state. Therefore, a better strategy is to selectively inhibit bacterial virulence, rather than to kill bacteria completely, in order to reduce the pressure to evolve resistance.
All known human pathogens require proteases, which are enzymes that break down proteins, to be infectious. This work focuses on a protease called ClpXP. The bacterium Caulobacter crescentus requires ClpXP when it invades a host as a pathogen, but does not need the protease for regular, non-virulent growth. ClpXP is therefore an ideal target for selective inhibition of bacterial virulence.
This study helps us understand how ClpXP selects certain proteins to break down at certain times. It elucidates the process by which various adaptors work together to select proteins and make them available to the ClpXP, so that ClpXP can destroy them. With this understanding, we can identify ways to block the process. We can also extend the findings in this particular case of C. crescentus to bacteria that pose grave threats to human health, such as Staphyloccus, Vibrio cholera, and Brucella abortus. In addition, because the ClpXP protease in the mitochondria of eukaryotic cells is very closely related to this system, we suspect that our work will inform the growing role of mitochondrial quality control in many human diseases, including cancer.
Read more about Peter Chien.
Associate Professor, Department of Food Science
Yang, T., Zhang, Z., Zhao, B., Hou, R., Kinchla, A., Clark, J. M., & He, L. (2016). Real-Time and in Situ Monitoring of Pesticide Penetration in Edible Leaves by Surface-Enhanced Raman Scattering Mapping. Analytical Chemistry, 88(10), 5243–5250. https://doi.org/10.1021/acs.analchem.6b00320
Chemical pesticides are widely used and play an essential role in agriculture production. Most chemical pesticides are designed to be toxic to living things, so by their very nature pose risks to human health and the environment. There is increasing evidence showing an association between pesticide exposures and human health problems, such as cancer and nervous system disorders. Fresh produce is a common source of pesticide exposure. Understanding the penetration of pesticides into fresh produce is particularly important, because pesticides within plant tissue are difficult to remove by rinsing compared to those on the plant surface. However, there is lack of an effective method that can measure pesticide penetration.
In this work, we developed an innovative method for monitoring pesticide penetration in spinach leaves. To perform the method, we apply gold nanoparticles to the surface of spinach leaves that contain pesticide. The gold nanoparticles penetrate quickly into plant tissues and complex with pesticide molecules, so that the nanoparticles go wherever there is pesticide. Then, we scan the spinach leaves using surface-enhanced Raman scattering (SERS) mapping. SERS is a spectroscopic technique that detects the location of pesticide clinging to gold nanoparticles. It gives us a depth map of pesticide penetration. This method has attracted the attention of BASF Corporation, which is using our technology to study new pesticides, in order to develop strategies to formulate and apply the pesticides safely and effectively.
Read more about Lili He.
Distinguished Professor, Department of Microbiology
Tan, Y., Adhikari, R. Y., Malvankar, N. S., Pi, S., Ward, J. E., Woodard, T. L., Nevin, K. P., Xia, Q.,Tuominen, M. T., and Lovley, D. R. (2016). Synthetic Biological Protein Nanowires with High Conductivity. Small, 12(33), 4481–4485. https://doi.org/10.1002/smll.201601112
Electrically conductive synthetic protein nanowires (e-SPNs) are a revolutionary electronic material. They are not only sustainably produced, but they have greater functionality than those made of traditional materials. This paper outlines a method of biologically producing e-SPNs, in which synthetic genes modify the electrically conductive protein filaments naturally produced by the microorganism Geobacter. While the native filaments have limited practical applications, the synthetic genes enabled Geobacter to produce copious quantities of e-SPNs that were not only thinner than the native filaments, but also much more conductive. Subsequent design of other synthetic genes has yielded highly functional wires that can sense specific chemicals or that have finely-tuned conductivity for specific types of sensors.
e-SPNs are well-suited for biomedical diagnostics and wearable or implantable nanowire devices for several reasons. They are more compatible with cells and tissues, and more stable in bodily fluids than standard silicon nanowires. Their adaptable functionalities enable the design of wearable electronic devices to monitor for a wide range of metabolic imbalances and disease. This research has established a new field of 'e-biologics', including a start-up company of that name, in which the power of synthetic biology can be harnessed to sustainably produce sophisticated electronic materials.
Read more about Derek Lovley.
Assistant Professor, Department of Veterinary and Animal Sciences
Wells, A. C., Daniels, K. A., Angelou, C. C., Fagerberg, E., Burnside, A. S., Markstein, M., Alfandari, D., Welsh, R. M., Pobezinskaya, E. L., and Pobezinsky, L. A. (2017). Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells. eLife, 6. https://doi.org/10.7554/elife.26398
The immune system is the only known natural mechanism that destroys infected or cancerous cells in an organism. Cytotoxic T lymphocytes, also called T-killer cells, are the most potent killer cells among all of the cells involved in the immune response to viruses and tumors. Unfortunately, under pathological conditions such as chronic viral infections or cancer, the function of T-killer cells is often compromised. When T-killer cells become inactive, it is very difficult to treat these diseases. The Pobezinsky lab is working to understand how to generate and maintain fully functional T-killer cells under pathological conditions.
This study reports the discovery of a molecular switch that turns T-killer cells on or off. The switch is based on certain RNA molecules called microRNA that regulate gene expression. The authors discovered that reducing the expression of a particular microRNA called let-7 results in enhanced T-killer cell function, and conversely, increasing the let-7 expression results in diminished T-killer cell function. Understanding this relationship opens up the possibility for designing therapies that decrease levels of let-7 in order to improve T-killer cell performance in responses to viruses and tumors. Dr. Pobezinsky recently received a large industry grant to directly explore these translational possibilities.
Read more about Leonid Pobezinsky.
Professor, Department of Chemistry
Mout, R., Ray, M., Yesilbag Tonga, G., Lee, Y.-W., Tay, T., Sasaki, K., & Rotello, V. M. (2017). Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing. ACS Nano, 11(3), 2452–2458. https://doi.org/10.1021/acsnano.6b07600
The recently developed gene editing tool CRISPR/Cas9 is a powerful technology with enormous potential to treat genetic diseases. CRISPR has two components: a scissor-like protein Cas9, and a RNA molecule called sgRNA that guides Cas9 protein to a target gene. Once the Cas9-sgRNA pair gets to the destination gene in the nucleus, it can find and correct the mistakes in the gene with the help of host cell repair machinery. Currently, CRISPR/Cas9 therapy is carried out by delivering genes to the host cells, so that the cells can make their own Cas9 and sgRNA. However, this strategy creates major problems of unwanted gene editing and immune responses because the CRISPR genes remain in the host cells after they are delivered.
This study demonstrates a highly efficient alternative nanomaterial-based strategy that directly delivers a pre-fabricated Cas9:sgRNA complex to the cells. It overcomes the challenges of crossing the cell membrane and arriving at the cell nucleus without being trapped in intracellular structures along the way. The Cas9:sgRNA complex was delivered to >90% of cells using a wide range of cell types. The strategy therefore provides a way to employ the powerful gene editing capabilities of the CRISPR system without suffering from its usual limitations. The research has been patented, and two industrial partnerships have been generated to apply our technology for therapeutic and agricultural use. Additionally, the technology has featured in multiple proposals to funding agencies.
Read more about Vincent Rotello.
Professor, Department of Chemistry
Dutta, K., Hu, D., Zhao, B., Ribbe, A. E., Zhuang, J., & Thayumanavan, S. (2017). Templated Self-Assembly of a Covalent Polymer Network for Intracellular Protein Delivery and Traceless Release. Journal of the American Chemical Society, 139(16), 5676–5679. https://doi.org/10.1021/jacs.7b01214
Protein-based drugs have great potential for improving our ability to treat disease. In comparison to current drugs that are based on small molecules, drugs composed of proteins are more effective for addressing specific genetic deficiencies without undesirable side effects. However, there are major challenges in delivering proteins into and within a cell. These challenges are related to keeping the protein stable, avoiding unwanted immune system response, and translocating the protein across the cellular membrane.
This study presents a novel and practical strategy which simultaneously overcomes all of these challenges. In this strategy, the polymers self-assemble to form a sheath around the protein, analogous to "shrink-wrapping" the protein. The polymer sheath encapsulates proteins, preserves their structure and function during delivery, and releases them when the assembly enters the cell cytosol. The polymers do not provoke an unwanted immune system response, and do not leaving any residue behind. This strategy is applicable to a broad range of proteins, and the sheath can be designed to release its cargo under various conditions. In addition to the applications for protein therapeutics and devices, the technology can be used to design reagents for basic biochemical research. Dr. Thayumanavan is currently working with industry partners toward both applications, in part through his start-up company, Cyta Therapeutics.
Read more about Sankaran Thayumanavan.
Professor, Department of Chemistry
Borotto, N. B., Zhou, Y., Hollingsworth, S. R., Hale, J. E., Graban, E. M., Vaughan, R. C., & Vachet, R. W. (2015). Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry. Analytical Chemistry, 87(20), 10627–10634. https://doi.org/10.1021/acs.analchem.5b03180
Protein therapeutics are the fastest growing segment of the pharmaceutical industry. Unlike traditional small‐molecule drugs, protein-based drugs must maintain not only their covalent structure (i.e. bonding of atoms in the molecule) but also their proper three‐dimensional (3D) structure. Changes in a protein therapeutic's 3D structure can lead to an inactive drug or worse, an unwanted immune response. Consequently, there is a big push in the pharmaceutical industry to find fast, reliable, and convenient methods to assess protein 3D structure.
While there are excellent traditional biochemical tools that can assess protein structure, they are either too slow to be routinely useful (e.g. NMR, X‐ray crystallography) or they are not sensitive enough to identify structural changes that influence the function of a protein therapeutic (e.g. circular dichroism (CD), fluorescence). Considering this gap in technology, we developed an approach based on chemical labeling and mass spectrometry (MS) that is more rapid and efficient than techniques like NMR and X‐ray crystallography and provides much more structural resolution than techniques such as CD and fluorescence.
The technique described in this paper is the subject of a pending patent, and a company called QuarryBio has recently licensed this technology. We are collaborating with QuarryBio on ways to further extend this method to make it more readily available for pharmaceutical companies to use.
Read more about Richard Vachet.
Assistant Professor, Department of Biochemistry and Molecular Biology
Pan, H., Oztas, O., Zhang, X., Wu, X., Stonoha, C., Wang, E., Wang, B., and Wang, D. (2016). A symbiotic SNARE protein generated by alternative termination of transcription. Nature Plants, 2(2), 15197. https://doi.org/10.1038/nplants.2015.197
Optimal food production by plants requires a sufficient supply of nitrogen. Historically, sustained agricultural productivity has been accompanied by increased application of synthetic nitrogen fertilizers. The chemical synthesis of nitrogen fertilizers is an energy-intensive process that consumes massive amounts of fossil fuel. At the same time, the application of nitrogen fertilizers is inefficient: plant roots can absorb only a small amount of nitrogen. The excess nitrogen fertilizer that is left in the soil causes a multitude of environmental issues. Therefore, new ways of supplying nitrogen to plants are needed to maintain a sustainable food supply without excessive environmental degradation.
Unique among crop species, legumes evolved a symbiosis with nitrogen-fixing bacteria and therefore require no nitrogen fertilizer. One long-standing goal of the research community is to gain sufficient understanding of the nitrogen-fixing symbiosis so as to expand the range of crops that can form this association. This study presents insight into how this symbiosis works. We found that the compartment housing the nitrogen-fixing bacteria, which is maintained by the host as an intracellular organelle, is defined by a specialized protein called SYP132A. We also discovered that SYP132A exists also in non-legume plants, where it is required for another, more ancient symbiosis with beneficial fungi, which is found in most land plants. This and other studies prove that certain basic architecture of the nitrogen-fixing symbiosis is already present in most non-legume crops. This insight will significantly simplify the task of engineering the nitrogen-fixing symbiosis in non-legume crops.
Read more about Dong Wang.