Richard Roberts - US grants

The Great Lakes Research Consortium (GLRC) proposes to immerse 20 undergraduate faculty in a 3-week summer practicum that demonstrates environmental problem-solving as an effective teaching strategy to stimulate undergraduates' interest in environmental science. This model has proven to be effective during a summer practicum for undergraduate environmental science teachers of two and four year colleges who, as a result of the practicum, successfully incorporated environmental problem solving curriculum into their courses. Undergraduate faculty participants learn environmental analysis techniques and prepare environmental impact statements (EIS) for a hypothetical development project in a contaminated harbor of Lake Ontario. As they are being exposed to new innovative theoretical concepts and techniques developed by the Great Lakes research community to understand and solve environmental problems, participants are shown how to integrate environmental problem-solving into curricula at their home institutions. Special topics, based on the Great Lakes experience, will include the theories and applications of cascading trophic interactions and particle-size spectra in community ecology; analytical methods for determining toxic chemical concentrations in sediments and fishes; and the use of microcomputers for massbalance and bioenergetics modeling of large lake systems. Through preparation of environmental impact statements for a realistic project, these techniques will be integrated into the overall environmental analysis and problem-solving approach that has stimulated undergraduate interest in science at two GLRC campuses for a decade. Participants in the practicum will return to their home institutions with expanded and updated professional skills and new strategies, methods and techniques for improving undergraduate education and addressing environmental problems in local communities.

[unreadable] DESCRIPTION (provided by applicant): There is a pressing need to develop new molecular tools that recognize protein surfaces for uses ranging from imaging probes to systems biology (blocking or modulating protein-protein interactions, serving as sensors) to proteomics (affinity reagents for mass spectrometry or chip-based diagnostics) to novel therapeutic leads. Here, we propose to implement and test a new combinatorial approach for constructing both natural and unnatural mRNA display libraries-PURE mRNA display (PURE = Protein synthesis Using Recombinant Elements). In this approach, the protein synthesis machinery will be assembled from purified components, providing complete experimental control of both concentrations and constituents. PURE mRNA display thus has several potential advantages relative to the classical mRNA display, most importantly in providing a facile route to construct designer display libraries bearing unnatural amino acids. Our specific aims are: 1. To test if it is possible to construct mRNA-peptide fusions using the PURE protein synthesis system. 2. To Compare and contrast PURE vs. retic-based mRNA display for in vitro directed evolution experiments. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Studying the localization of proteins with conventional antibodies has greatly contributed to our understanding of the structure and function of neurons. However, conventional antibodies have several limitations that drastically limit their utility. Tissue must be fixed and permeabilized prior to staining and often the overlapping expression patterns of adjacent neurons are difficult to interpret because of the lack of contextual information. For these reasons, the precise subcellular localization patterns in vivo of the majority of neuronal proteins have not been well characterized. The purpose of the studies proposed in this grant is to develop genetically encoded probes that will allow the subcellular localization of neuronal proteins to be mapped in vivo and in real time with high fidelity. These probes consist of genetically encoded aptamers (intrabodies) that bind to endogenous neuronal proteins and are generated using the mRNA display system. Three different types of intrabodies will be generated: 1. Binders to individual cytoskeletal proteins that mark neuronal structures such as pre- and postsynaptic sites. 2. Binders to transmembrane proteins. These intrabodies will be modified to enable them to label either total protein or only protein that is present on the plasma membrane of the cell. 3. Binders to activated G-proteins. Intrabodies will be used to attach three types of molecules to endogenous target proteins: 1. Fluorescent molecules that can be used to report the localization of the protein. 2. proteins for measuring Ca++ concentration in the region around the protein. 3. proteins that are activated by light to produce depolarizing currents. Subcellular trafficking of proteins is crucial to virtually all neuronal functions, including establishment of synaptic connections, axon guidance and synaptic plasticity. Disruption of protein trafficking has been linked to such diseases as Alzheimer's disease and Parkinson's disease. Protein trafficking also plays a critical role in drug addiction. Intrabodies generated through RNA display will provide tools to map the subcellular localization of endogenous proteins with high fidelity, in vivo and in real time, which is not possible with current technology.

DESCRIPTION (provided by applicant): In this project, we propose to combine mRNA display (a protein design/evolution method) and high efficiency microfluidic sorting to create a new technology-microfluidic mRNA display-for the purpose of enabling design of peptides and proteins that can be used as protein capture reagents. We will develop and apply this powerful new technology toward creating a comprehensive reagent set aimed at the Hepatitis C virus (HCV) proteome. In this section, we begin by describing the existing state-of-the-art in 1) mRNA display-based peptide and protein design, 2) bead-based micromagnetic separations, and 3) give an introduction to the proteins expressed by the HCV that will be the targets of this work. This is followed by our description of how we will integrate these technologies to achieve our goal of high-throughput development of new protein capture reagents.

DESCRIPTION (provided by applicant): The primary goal of this proposal is to increase the pace of experimental determination of the function of large and high priority gene families in bacterial genomes. These genes can help elucidate the mechanisms of antibiotic resistance, provide new drug targets or improve our understanding of the human microbiome. Specifically, we propose to catalyze the formation of a consortium of experimental and computational biologists that would collaborate directly to test experimentally the predicted functions of high-priority genes of currently unknown function or specificity. Central to this effort would be the creation of a community web-based database (portal) which would allow computational and experimental scientists to communicate easily, assist experimentalists in identifying those high-priority genes for which there are the highest- quality computational predictions for their molecular function, and providing feedback to the computational biologists, since it remains true that the insights and experience of the dedicated biochemist can be essential in guiding the development of algorithmic sophistication. Experimental validations of gene function would be reported in a manuscript when successful, or as annotations in the prediction database when negative. Many existing groups, both large and small, have the relevant expertise and could contribute to the overall effort by performing the pertinent gene function determination studies. During the course of the project it is anticipated that at least 100 gene families will be identified and subjected to experimental tests, directly affecting the annotations of thousands of important genes. The initial project will fund 40 teams across the US, and will create more than 40 new jobs providing a significant economic stimulus. It will also stream-line and integrate the process of computational predictions and biochemical function validation leading to significant improvement in the cost of future work. In addition to direct benefits to microbial biology, infectious disease research and computational biology this public experiment in the form of a new social network might have long-term transformative implications for funding and other economic implications. PUBLIC HEALTH RELEVANCE: This project will be important in providing fundamental knowledge to many aspects of infectious disease research. Unknown genes from several important pathogens such as Mycobacterium tuberculosis and Helicobacter pylori will be high priority targets as will genes in model organisms with orthologous genes that are widely distributed in both bacterial pathogens and higher organisms including humans. A key factor in deciding priorities will be the health implications of successful predictions.

DESCRIPTION (provided by applicant): This application aims to demonstrate that Scanning Unnatural Protease Resistant (SUPR) peptides provide a general solution to the problem of targeting traditionally undruggable proteins. To do this, we will use mRNA display with an expanded genetic code to create a new class highly stabilized, membrane-permeant peptides that can block or modulate protein-protein interactions for two of the most important intracellular proteins conferring the oncogenic phenotype-the activated form of Ras and the Stat3 protein. Our three Specific Aims are: 1) To design stabilized SUPR peptides targeting intracellular undruggable proteins involved in cancer transformation or maintenance, 2) To characterize and enhance selected SUPR peptide functions towards cancer drug applications, and 3) To evaluate in vivo characteristics and assess the therapeutic potential of optimized SUPR peptide drug candidates for cancer treatment in mice. Overall, this project is intended to develop novel molecules as well as a general approach to target cancer-relevant proteins that have proved challenging up to this point-so much so that the proteins may be called undruggable.

Diagnosing and treating cancer requires having a reliable set of affinity reagents that can specifically and strongly bind to cancer-related protein targets. These reagents are the necessary molecular tools that will enable next-generation technologies for studying, diagnosing, and fighting cancer. Current approaches to producing such reagents, however, are unreliable, expensive, and slow. Existing reagent generation methods (e.g., hybridoma technology, phage display, yeast display) are not readily adapted to leverage high throughput library sequencing. Where reagents do exist, they are often both extremely expensive and poorly characterized. It is these important shortfalls that this research project aims to address. This project combines two powerful existing technologies: 1) mRNA display and 2) Modular Microfluidic and Instrumentation Components (MFIC) to dramatically speed target-directed, renewable reagent development. mRNA display is a molecular selection technology that is uniquely capable of searching libraries of more than a trillion unique compounds to develop ultrahigh affinity reagents against cancer-relevant targets. While this technology has an impressive demonstrated track record of producing such reagents, it has so far been limited to the laboratory scale. This project will adapt it to a true high-throughput format by integrating it into a continous flow microfluidic system based on MFIC technology. By automating mRNA display, this project will make it broadly accessible to the research community while decreasing its cost and increasing its throughput. Once the automated system is developed, we will use it to produce affinity reagents that target two key cancer screening protein markers?PSA and CA125. These markers are broadly used (they were the primary tools used in 154,000 patient Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)) and renewable, inexpensive reagents that recognize these biomarkers would clearly be of utility. An automated microfluidic mRNA display system will be used to develop novel, specific, and high affinity reagents for these targets. Developing a microfluidic approach for mRNA display selection will involve implementing a magnetophoretic separation system that can both perform affinity selections and purify the products of preparative biochemical reactions. An automated system for preparing mRNA reagents will be combined with an automated target selection system. The DNA-encoded products of this selection will be amplified in a microfluidic PCR system. The entire process workflow will be implemented in a closed loop to enable multiple rounds of selection and amplication, producing an optimal high affinity binding reagent. Each step of the automated mRNA display will be benchmarked for quality assurance by developing affinity reagents for the cancer marker Bcl-xL; standard manual mRNA display has proved resoundingly successful at producing such reagents and there is plentiful existing data against which to benchmark.