2005 CCG Fisher Pilot Project Awardees

University of Michigan Life Sciences Institute's collaboration with Fisher Scientific International Inc. has awarded its first grants to UM researchers under the Center for Chemical Genomics Pilot Project Initiative. The funds will provide research support for up to two years, allowing University of Michigan scientists to conduct innovative research directed toward technology development relating to high throughput screening, small molecules synthesis, target detection and assay miniaturization.

Researchers receiving awards are

• Associate Professor of Electrical Engineering Jay Guo for "Label-free Optical Micro-Resonator Biosensor for High-throughput Screening Applications";
• Life Sciences Institute Research Assistant Professor and Molecular, Cellular and Developmental Biology Assistant Professor Anuj Kumar for "High-Throughput FRET Screening in Yeast with Plasmid-Based FP Fusions";
• Assistant Professor of Biomedical Engineering and Chemical Engineering Michael Mayer for "Efficient and Parallel Formation of Membrane Protein Arrays by Hydrogel-based Microcontact Printing";
• and Professor of Chemistry John Montgomery for "New Methods for Macrocycle Glycosylation."

Abstracts

L. Jay Guo, Ph.D.: "Label-free Optical Micro-Resonator Biosensor for High-throughput Screening Applications"

Affiliations:
Associate Professor, Electrical Engineering & Computer Science
Associate Professor, Macromolecular Science & Engineering
Associate Professor, Applied Physics

Highly sensitive optical detectors for biomolecules that can be easily multiplexed to form sensor arrays are excellent candidates for high-throughput screening applications. Eliminating the use of fluorescently tags on the analyte molecules will further maintain the bio-functionality of the protein molecules and thereby providing more reliable data on the protein binding kinetics.

We propose to develop a novel optical system for studying real-time protein binding interactions and for high-throughput screening of chemical compounds that are important in protein-protein interactions. The method is based on optical microresonators (OMR) of very high quality-factor acting as highly sensitive reporter of biomolecular binding events. These microresonators, designed with integrated optics techniques, are formed using ring type shaped waveguides. A typical dimension of such a microresonator is 20 m m to 100 m m depending on the optical wavelength and other design parameters. Our preliminary measurements showed a high sensitivity for detecting glucose concentration and biotin-streptavidin binding. These experiments were performed using microring resonators fabricated by batch-processing technique and of moderate quality factor (Q=1000-10,000). These results imply that OMR sensor arrays have the potential of providing high-throughput analysis of biomolecular interactions without fluorescently tagging the analyte molecules.

To develop this new biosensor technology several technical and scientific problems must be addressed. This pilot program aims to investigate the OMR detection limits for both proteins and small compound molecules; by adopting surface modification techniques to generate receptor binding sites, we will screen model protein systems and establish sensor's dynamic range, linearity and repeatability; we will design integrated optics elements and construct sensor probes that are compatible with 96-well format, and also optimize the OMR sensor by using novel waveguide geometry to enhance the device sensitivity.

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Anuj Kumar, Ph.D.: "High-Throughput FRET Screening in Yeast with Plasmid-Based FP Fusions"

Affiliations:
Assistant Professor, Dept. of Molecular, Cellular and Developmental Biology
Research Assistant Professor, Life Sciences Institute

This grant application proposes to pioneer the development of methods for high-throughput screening of protein-protein interactions in yeast by fluorescence resonance energy transfer (FRET). Specifically, we will:

1. Construct a plasmid-based collection of yeast promoter-ORF-fluorescent protein fusions as "moveable" genes. By a recombination-based approach, a set of 484 yeast genes with native promoters will be cloned as C-terminal fusions to fluorescent proteins (FPs). This gene set encompasses all known yeast kinases and transcription factors, as well as known components of selected protein complexes (Aim 2). In addition to being tagged with either YFP or CFP, each yeast promoter-ORF will be flanked by viral att sites for easy transfer to any vector containing compatible att termini. The FP fusions, therefore, are both appropriate reagents for live cell fluorescence microscopy and a resource of moveable yeast genes with native promoters—a unique and widely useful resource for the yeast scientific community.
2. Establish methods for screening protein interactions by FRET. The plasmid-based FP fusions described above will be used to screen for protein interactions by FRET. Methods for highthroughput FRET analysis will be established through the study of proteins involved in the formation of clathrin-coated vesicles at the trans-Golgi network in yeast; these methods will then be used to screen larger sets of putative protein interactions. This high-throughput FRET analysis will constitute the first such study in any eukaryote, complementing existing two-hybrid screens and co-IP/mass spectrometry approaches.

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Michael Mayer, Ph.D.: "Efficient and Parallel Formation of Membrane Protein Arrays by Hydrogel-based Microcontact Printing"

Affiliations:
Assistant Professor, Biomedical Engineering
Assistant Professor, Chemical Engineering

We propose a highly efficient and cost-effective method to fabricate functional arrays of membrane proteins using hydrogel stamping. In this method we will use a topographically patterned agarose gel to create microspots of bilayers containing many different membrane proteins, which are functionally-embedded in fluid lipid bilayers. The capability of agarose gels to store biomolecules (in this case, lipids and transmembrane proteins) in a hydrated and biocompatible environment will allow us to create rapidly many copies of arrays of membrane proteins with minimal consumption of these extremely fragile and precious biomolecules. We will explore the potential of the resulting arrays for high-throughput screening for potential drug candidates by exposing the arrays to agonists and antagonists of G protein coupled receptor and ion channel proteins such as the HERG channel.

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John Montgomery: "New Methods for Macrocycle Glycosylation"

Affiliation: Professor, Department of Chemistry

The underlying principal objective of our research program is the development of new synthetic procedures, typically involving transition metal catalysis, that improve the selectivity and efficiency by which complex, biologically interesting molecules may be constructed. A recent achievement of our group was the development of a new catalytic procedure for the construction of complex macrocyclic structures ( J. Am. Chem. Soc. 2005, 127, 13156). The principal aim of this proposal is to devise a new method for the glycosylation of the complex macrocyclic structures that may be constructed by this new technology. Our approach will involve development of a two-step method for the assembly and stereoselective glycosylation of complex macrocycles, potentially without protecting groups. The exquisitely selective molecular recognition imparted by carbohydrate units, coupled with the substantial structural diversity in complex molecules that our nickel cyclization technology allows, provides significant opportunities for the preparation of glycoconjugates with a broad array of biological properties. The potentially rapid and efficient access to complex glycoconjugates of unnatural macrocyclic structures will allow these diverse structures to be examined in a number of problems in chemical biology.

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