Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ~30 sec time resolution. The entire library is kinetically screened against a single probe in ~14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.
We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.
We describe a new oblique-incidence reflectivity difference (OI-RD) scanning microscope for high-throughput
screening, in microarray format on functionalized glass slides, small-molecule compound libraries for protein ligands.
The microscope employs a combination of scan mirror for y-scan and single-axis translation stage for x-scan. For a
printed microarray with over 10,000 features, each of 100 μm in diameter and distinct small molecule targets, we can
acquire an end-point image of the microarray in 90 minutes with pixel resolution of 20 μm × 20 μm. The microscope is
also capable of measuring binding kinetics of over 10,000
protein-ligand reactions simultaneously. We also describe a
number of strategies for immobilizing small molecule compounds on functionalized glass slides: (1) conjugating the
compounds (through a chemically inert linker) with a lysine residue so that the primary amine on the lysine serves as the
anchor to epoxy-functionalized glass surface; (2) conjugating the compounds (through a linker) with a biotin residue so
that the biotin serves as the anchor to streptavidin-functionalized glass surface; (3) immobilizing small molecule
compounds without modification on isocyanate-functionalized glass surface through non-specific reaction of
nucleophilic molecular motifs on most bioactive compounds with isocyanate groups. We present preliminary
measurements of protein-small molecule binding reactions using the new microscope and the surface immobilization
strategies.
Small-molecule microarrays composed of tens of thousands of distinct synthetic molecules, natural products, and their
combinations/modifications provide a high-throughput platform for studying protein-ligand interactions. Immobilization
of small molecule compounds on solid supports remains a challenge as widely varied small molecules generally lack
unique chemical groups that readily react with singly or even multiply functionalized solid support. We explored two
strategies for immobilizing small molecule compounds on
epoxy-functionalized glass surface using primary-aminecontaining
macromolecular scaffolds: bovine serum albumin (BSA) and
amine-modified poly-vinyl alcohol (PVA).
Small molecules with N-hydroxysuccinimide (NHS) groups were conjugated to BSA or amine-modified PVA.
Small-molecule-BSA conjugates and small-molecule-PVA conjugates were subsequently immobilized on epoxy-functionalized
glass slides through amine-epoxy reactions. Using an
oblique-incidence reflectivity difference (OI-RD) scanning
microscope as a label-free detector, we performed a comparative study of the effectiveness of BSA and PVA as
macromolecular scaffolds for anchoring small molecule compounds in terms of conjugation efficiency, surface
immobilization efficiency, effect of the scaffold on end-point and kinetics of subsequent binding reactions with protein
probes.
In this study we report new results on the changes in optical properties of glass surfaces induced during wet photolithography using Oblique Incidence Reflectivity Difference Ellipsometry (OI-RD). A novel wet UV-photolithographic method for patterning phospholipid bilayers into two-dimensional arrays of voids and patches on hydrophilic glass substrates is presented. Especially, this technique involves etching the glass substrate fused with lipid vesicle solution and subsequently illuminated with short-wavelength UV light through a photo mask thus creating voids in the irradiated regions. The effects of the chemical etching and subsequent UV irradiation on the surfaces of microscope glass slides are investigated using the OI-RD technique. In this study, we have observed that the UV irradiation after chemical etching further changes the properties of the surface, even in the absence of the lipid bilayer. As a result, irradiating the chemically etched surface before the UV photolithography step renders the whole surface homogenous. Furthermore, fluorescence recovery after photobleaching (FRAP) experiments have been conducted on such homogenized surfaces which reveal that the fluidic properties of the membranes are retained. The created patterns are suitable to study protein-DNA interactions in the lipid environment. Our long term goal is to utilize this technique as a new screening approach for testing drug interactions above and below the cell surface.
We developed an optical oblique-incidence reflectivity difference (OI-RD) scanning microscope for imaging microarrays of label-free protein and DNA. Such a microscope complements currently widely used fluorescence-based optical microscopes by offering the capability to detect biochemical activities of DNA and protein molecules without the influence of fluorescent-labeling molecules. The specific activity and function of protein molecules are particularly subject to binding of small or large foreign molecules either directly through conformational change in the protein molecule itself or indirectly through properties of the attached molecules. We show that an OI-RD microscope can be used to: 1) detect binding reactions on microarrays without labeling, 2) quantitatively measure the optical properties of microarray spots, and 3) detect microarrays submerged in solution (which will enable OI-RD to monitor reaction kinetics on microarrays in reactive solutions). Furthermore, using both OI-RD and fluorescence images of an immunoglobulin-G (IgG) protein microarray, we observed that labeled and unlabeled IgG molecules deposited on the microarray substrate exhibit different wetting behaviors, and a mixture of the two tends to segregate into labeled and unlabeled regions. This illustrates potentially undesirable effects of fluorescent-labeling agents on protein properties that are of interest.
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