BACKGROUND: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function. The methods used to screen natural products are not feasible for large libraries composed of covalently immobilized compounds on synthesis beads. The sheer number of compounds synthesized by split-pool synthesis, and the small quantity of individual compound attached to each bead require assay miniaturization for efficient screening. RESULTS: We present a miniaturized cell-based technique for the screening of ligands prepared by split-pool synthesis. Spatially defined droplets with uniform volumes of approximately 50-150 nanoliters (depending on well dimensions) are arrayed on plastic devices prepared using a combination of photolithography and polymer molding. Using this microtechnology, approximately 6,500 assays using either yeast cells or mammalian tissue culture can be performed within the dimensions of a standard 10 cm petri dish. We demonstrate that the biological effect of a small molecule prepared by split-pool synthesis can be detected in this format following its photorelease from a bead. CONCLUSIONS: The miniaturized format described here allows uniformly sized nanodroplets to be arrayed on plastic devices. The design is amenable to a large number of biological assays and the spatially arrayed format ensures uniform and controlled ligand concentrations and should facilitate automation of assays. The screening method presented here provides an efficient means of rapidly screening large numbers of ligands made by split-pool synthesis in both yeast and mammalian cells.
BACKGROUND: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function. The methods used to screen natural products are not feasible for large libraries composed of covalently immobilized compounds on synthesis beads. The sheer number of compounds synthesized by split-pool synthesis, and the small quantity of individual compound attached to each bead require assay miniaturization for efficient screening. RESULTS: We present a miniaturized cell-based technique for the screening of ligands prepared by split-pool synthesis. Spatially defined droplets with uniform volumes of approximately 50-150 nanoliters (depending on well dimensions) are arrayed on plastic devices prepared using a combination of photolithography and polymer molding. Using this microtechnology, approximately 6,500 assays using either yeast cells or mammalian tissue culture can be performed within the dimensions of a standard 10 cm petri dish. We demonstrate that the biological effect of a small molecule prepared by split-pool synthesis can be detected in this format following its photorelease from a bead. CONCLUSIONS: The miniaturized format described here allows uniformly sized nanodroplets to be arrayed on plastic devices. The design is amenable to a large number of biological assays and the spatially arrayed format ensures uniform and controlled ligand concentrations and should facilitate automation of assays. The screening method presented here provides an efficient means of rapidly screening large numbers of ligands made by split-pool synthesis in both yeast and mammalian cells.
Authors: K M Sakamoto; K B Kim; A Kumagai; F Mercurio; C M Crews; R J Deshaies Journal: Proc Natl Acad Sci U S A Date: 2001-07-03 Impact factor: 11.205
Authors: Wesley G Cochrane; Marie L Malone; Vuong Q Dang; Valerie Cavett; Alexander L Satz; Brian M Paegel Journal: ACS Comb Sci Date: 2019-03-29 Impact factor: 3.784
Authors: Paul Sirajuddin; Sudeep Das; Lymor Ringer; Olga C Rodriguez; Angiela Sivakumar; Yi-Chien Lee; Aykut Üren; Stanley T Fricke; Brian Rood; Alpay Ozcan; Sean S Wang; Sana Karam; Venkata Yenugonda; Patricia Salinas; Emanuel Petricoin; Michael Pishvaian; Michael P Lisanti; Yue Wang; Richard Schlegel; Bahram Moasser; Chris Albanese Journal: Cell Cycle Date: 2012-09-14 Impact factor: 4.534