| X-ray crystallography is arguably the most powerful experimental tool to determine high-resolution structures of biomacromolecules. In protein crystallography, a popular methodology named multiwavelength anomalous dispersion (MAD) requires the incorporation of selenomethionine during peptide biosynthesis. However, no equivalent methodology was developed for nucleic acids prior to this work.; In our studies, new nucleoside analogues containing selenium functionalities at 2', 3', and 5' positions were chemically synthesized. Some of these modified nucleoside monomers were incorporated site-specifically to oligonucleotides through phosphoramidite chemistry. Similar to selenomethionine derivatization in protein X-ray crystallography, selenium-derivatized nucleosides may be used for X-ray crystal structure determination of nucleic acids and nucleic acid-protein complexes, by the MAD method.; For the first time, we have successfully demonstrated this new strategy by covalently derivatizing nucleotides with selenium at the 2' -position for phase and structure determination. The successful incorporation and the stability of selenium functional group were verified by NMR, mass spectrometry, X-ray fluorescence and X-ray crystallography. Experimental results show the 2'-deoxy-methylselenouridine residue displays C3'-endo conformation in oligonucleotides, which is characteristic of RNA and A-form DNA double helix. Moreover, this new method provides a rational way to make isomorphous nucleic acid crystals for X-ray crystallography, which was carried out previously by heavy metal soaking. |
