Construction of Photo-Cross-Linked Microarrays of Small Molecules
Small molecule microarrays are one of the most promising approaches to screen ligand molecules for individual proteins of interest. However, their potential has not been fully realized due to the limited number of methods to introduce small molecules onto
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Abbreviations DSC DIPEA DMF rt UV
N,N′-disuccinimidyl carbonate N,N-diisopropylethylamine N,N-dimethylformamide Room temperature Ultraviolet light
1. Introduction Construction of small molecule microarrays has mainly been accomplished using the selective coupling approach. That is, a library of small molecules having a certain functional group (e.g., thiol) is printed onto solid surfaces derivatized with a different functional group (such as maleimide) that reacts selectively with the former one. Thus, the library molecules readily attach to the surface through a coupling reaction (1). However, to construct small molecule microarrays by using this protocol, one must synthesize a library of compounds having a certain functional group, Mahesh Uttamchandani and Shao Q. Yao (eds.), Small Molecule Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 669, DOI 10.1007/978-1-60761-845-4_2, © Springer Science+Business Media, LLC 2010
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or select such compounds from an in-house library. Natural product libraries, which usually consist of a range of different types of molecules, are therefore considered to have poor compatibility with this platform. However, a method having high compatibility with functional groups would avoid this problem. There is another drawback in using the selective coupling approach. Introduction of a tether, which connects a small molecule with a solid surface, on a defined site of a small molecule reduces the number of binding modes available for each compound. A decrease in the number of binding modes poses a major drawback to ligand screening and chemical genomics, especially when the goal is to discover new interactions between small molecules and proteins of interest. To overcome these drawbacks of the selective coupling approach, we developed a unique “non-selective” photo-crosslinking protocol for immobilizing a variety of small molecules, including natural products, on glass slides (2, 3).
2. Materials 2.1. Preparation of Photo-Cross-LinkerCoated Glass Slides
1. Amine-coated glass slide: HA-coated slide glass for DNA microarray (Matsunami Glass Industries, Ltd., Osaka, Japan). 2. Gap cover glass (Matsunami Glass Industries, Ltd., Osaka, Japan). 3. Slide staining chamber (BT-220; Matsunami Glass Ind., Ltd., Osaka, Japan). 4. MildMixer XR-36 instrument (Taitec Co., Ltd., Saitama, Japan). 5. Spin Dryer Mini (Wakenyaku Co., Ltd., Kyoto, Japan). 6. Slide activation solution: Prepare a solution of N,N ′-disuccinimidyl carbonate (100 mM) and N,N-diisopropylethylamine (100 mM) in reagent-grade N,N-dimethylformamide (DMF) prior to use. 7. Reagent grade ethanol. 8. Milli-Q water. 9. Photo-cross-linker solution: Prepare a solution of photo-crosslinker (compound 1 in Fig. 1, see Note 1) (100 mM) and N,Ndiisopropylethylamine (500 mM) in reagent-grade DMF. 10. Blocking solution: 2-ethanolamine (1 M) in reagent-grade DMF.
2.2. Small Molecule Printing and Immobilization
1. Library of small molecules. 2. 384-well polypropylene plate (X6004; Genetix LTD., New Milton,
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