We propose to develop a technique for mapping post-translational chemical modifications of histones in chromatin by direct imaging of single molecules. Our approach is to use a new imaging mode for the atomic force microscope (AFM) that allows chemical composition to be read directly and at the same time as a high resolution molecular image is acquired. In this way, we will observe both the pattern of post-translational modifications, such as acetylation, and the consequent local modifications of chromatin structure. The mode requires that a cognate ligand be tethered to the AFM probe. Antibodies have not proved adequate for this purpose, so we propose to develop synthetic ligands that are (a) highly reproducible (b) chemically simple and stable and (c) capable of recognizing small chemical modifications. Specifically, we propose to: 1. Develop DNA aptamers that are highly specific for acetylated histones. 2. Test the new aptamers in the simultaneous topography and chemical imaging mode of the AFM ('recognition imaging') using artificial acetylated histone H4 arrays developed in the Peterson lab at the University of Massachusetts Medical School. 3. Carry out an initial recognition imaging study of promoter chromatin extracted from a mouse cell line at the Georgel lab at Marshall University. The proposed work will pave the way for a more ambitious project aimed at analyzing chromatin from one (or a few cells) by exploiting the very small sample requirements of atomic force microscopy. ? ? ?
| Yu, Hanyang; Jiang, Bing; Chaput, John C (2011) Aptamers can discriminate alkaline proteins with high specificity. Chembiochem 12:2659-66 |
| Lin, Liyun; Fu, Qiang; Williams, Berea A R et al. (2009) Recognition imaging of acetylated chromatin using a DNA aptamer. Biophys J 97:1804-7 |
| Williams, Berea A R; Lin, Liyun; Lindsay, Stuart M et al. (2009) Evolution of a histone H4-K16 acetyl-specific DNA aptamer. J Am Chem Soc 131:6330-1 |
| Lin, Liyun; Hom, Doris; Lindsay, Stuart M et al. (2007) In vitro selection of histone H4 aptamers for recognition imaging microscopy. J Am Chem Soc 129:14568-9 |
