CHARACTERIZATION AND APPLICATION OF OSMOTIC DEWATERING TO THE CRYSTALLIZATION OF OLIGONUCLEOTIDES

The recent increase in research and applications of oligonucleotides i n new drug design has stimulated a need to determine the three-dimensi onal structures of oligonucleotides and their complexes with various l igands. Three-dimensional structure determination relies on X-ray diff raction data from high-quality crystals. However, only a very limited number of RNA molecules have been crystallized, and a broad record of experience in oligonucleotide crystallization is lacking. Osmotic dewa tering is a crystallization method in which the oligonucleotide and it s precipitant are concentrated at a controlled rate by the removal of water through a reverse-osmosis membrane using a specified concentrati on gradient. Therefore, rates of nucleation and crystal growth can be controlled. In this study, a transport model was developed to predict the water removal rates at various concentration gradients. Experiment al dewatering rate data agreed very well with theoretical calculations . Model predictions were applied to different osmotic dewatering cryst allization devices to crystalize a model RNA, ''U-U dodecamer''. High- quality dodecamer crystals were successfully grown and yielded a maxim um X-ray diffraction resolution of 2.46 Angstrom and usable resolution of 3.0 Angstrom. The results of this research will facilitate the app lications of osmotic dewatering to crystallographic studies and indust rial purification of biopharmaceuticals.