Enzymatic Synthesis of Phosphoroselenoate DNA Using Thymidine 5'-(α-P-seleno)triphosphate and DNA Polymerase for X-ray Crystallography via MAD

摘要

X-ray crystallography is the most direct and powerful tool for 3-D structure determination of biomacromolecules, such as ribozymes, viral RNA, and RNA-protein and DNA-protein complexes. This technology has significantly facilitated studies of macromolecular functions and mechanisms at the atomic level. Besides the difficulties related to crystallization, however, heavy atom derivatization for phase determination, a limiting factor in nucleic acid X-ray crystallography, has slowed determination of new structures. To facilitate phase and structure determination, the multiwavelength anomalous dispersion (MAD) technique has been developed for proteins via replacement of sulfur with selenium in methionine residues, which approximately accounts for two-thirds of all new protein structures. Derivatization with bromine on position 5 of pyrimidines and the RNA-binding protein U1A complex have also been used in MAD phasing of DNAs and RNAs. In comparison to those for protein, however, derivatization tools in X-ray crystallography for nucleic acids are quite limited.