Zhen Huang, Ph.D.
Department of Chemistry
Brooklyn College
2900 Bedford Ave., Brooklyn, NY 11210
Phone: (718) 951-5746, Fax: (718) 951-4607
E-mail: [email protected]
Determination of the three-dimensional structures of RNA molecules, RNA-protein and DNA-protein complexes with high resolution is invaluable for gaining understanding of biological systems at the molecular level. X-ray crystallography is the most direct and powerful tool for structure determination of these macromolecules. However, derivatization with heavy atoms for phase determination, a long-standing problem in nucleic acid X-ray crystallography, has slowed down the structural determination process. It can take years just to prepare derivatives and to determine the required phase information.
Recently, we have successfully demonstrated a novel derivatization strategy via selenium replacement of nucleotide oxygen.1-3 Unlike conventional halogen derivatization (Br or I), where halogens are primarily placed on the 5-position of deoxyuridine (a mimic of thymidine), selenium can be selectively introduced to a variety of positions via oxygen replacement (e.g., 2'-, 3'-, 5'-ribose oxygen, furan ring oxygen, non-bridging phosphate oxygen, or oxygen on nucleobases). Choice of positioning can avoid disruption of structure and function caused by modification. As the Multiwavelength Anomalous Dispersion (MAD) signal of selenium is as strong as that of bromine, selenium MAD phasing can be an alternative to the current bromine MAD phasing in nucleotide X-ray crystallography.
Our research results have shown that diffraction quality crystals of the 2'-selenium-derivatized oligonucleotides, such as 5'-GUSeGTACAC-3' (Se-octamer containing 2'-Me-Se-uridine) and 5'-GCGTAUSeACGC-3' (Se-decamer containing 2'-Me-Se-uridine), were identified, and X-ray fluorescence spectra confirmed the presence of selenium in crystals.2 MAD data of the Se-decamer to 1.2 Å resolution were collected and the diffraction data were successfully phased on the basis of the selenium anomalous signal.3 Likewise, diffraction data of the octamer to 1.8 Å resolution were collected, and the structure of the octamer was determined by the molecular replacement technique. These X-ray crystal structures also confirmed the presence of the 2'-methylseleno group at the a-position of the uridine.
In both structures, the 2'-Me-Se-substituted furanoses display C3'-endo puckers, consistent with the A-form geometry of the unmodified decamer and octamer duplexes, which is adopted by RNA and A-form DNA. As previously established for 2'-O-methylated nucleotides and other 2'-O-modified ribonucleotide analogs, the methyl groups of the methylseleno moieties are directed into the minor groove and the C3'-C2'-Se-Me torsion angles adopt an antiperiplanar conformation. As the 2'-a-position selenium derivatization retains the native C3'-endo conformation of A-Form DNA and RNA molecules, this 2'-selenium label approach is suitable for RNA and A-Form DNA derivatization for X-ray crystallography. So far, our progress in this project of selenium derivation on nucleic acids has demonstrated for the first time a new strategy to covalently derivatize nucleotides with selenium for phase and structure determination in X-ray crystallography, and have shown that the MAD phasing technique works with this selenium derivatization strategy.
As one selenium atom enables phase determination for RNAs or DNAs up to 30 nt. (based on an approximate calculation of X-ray phasing power) and the frequency of a building block in a nucleotide sequence is high (25% average rate), selenium-labeled dU (mimic of T) and U building blocks should meet most of the needs for DNA and RNA derivatization. Here we present you a product that can serve as both selenium-labeled dU and U building blocks for RNA and DNA Derivatization in X-ray Crystallography.
2'-Se-Me-U-CE Phosphoramidite has been discontinued
