The high-resolution structure of the DNA (5′-GTGTACA-C-3′) with the selenium derivatization at the 2′-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 Å resolution) with the 2′-Se modification in the minor groove is isomorphorous to the native structure (2.0 Å). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 Å resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.
We have developed a route for the synthesis of 2'-selenium uridine analogues and oligonucleotides containing selenium labels, and have demonstrated for the first time a new strategy to covalently derivatize nucleotides with selenium for phase and structure determination in X-ray crystallography.
We report here the first study of enzymatic synthesis of two phosphoroselenoate (PSe) DNAs using the two alpha-Se-TTP diastereomers (Sp and Rp) and DNA polymerase. The experimental results indicate that Klenow equally recognizes the two individual diastereomers at the same level as natural TTP. The incorporations of the PSe groups at the expected sites have been confirmed by the digestion resistance to exonuclease III, and the different patterns of the digestion resistance of DNA I and II indicate the configurational differences of the PSe centers (Sp or Rp). Unlike chemical synthesis, which is limited to short DNAs and where the separation of the PSe DNA diastereomers is necessary, this enzymatic method can be used to prepare longer DNAs without diastereomer separation. This quantitative enzymatic approach is particular valuable for the synthesis of longer DNAs with multiple PSe groups in large scale for their X-ray crystal structure determination by the MAD phasing technique.
The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.
We report here the synthesis of nucleoside and oligonucleotide analogs containing selenium, which serves as an anomalous scattering center to enable MAD phase determination in nucleotide X-ray crystallography. We have developed a phase transfer approach to introduce the selenium functionality in A, C, G, T, and U nucleosides at 5'-positions. In the incorporation of the selenium functionality, the leaving groups (bromide, mesyl, and tosyl) were readily displaced by sodium selenide, sodium diselenide, and sodium methyl selenide with yields higher than 90%. Selenium-derivatized oligonucleotides have been synthesized via phosphoramidite chemistry.
We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2'-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2'-methylseleno cytidine phosphoramidite and improved the accessibility of the 2'-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2'-selenium derivatization had no effect on the stability of the duplexes with the 3'-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3'-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2'-Se-C and 2'-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2'-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.
As oxygen and selenium are in the same group (Family VI) in the Periodic Table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucleoside 5'-(α-P-seleno)triphosphates (NTPαSe, including A, C, G, and U). We found that T7 RNA polymerase recognizes NTP SeαSp diastereomers as well as the natural NTPs, while NTPαSe Rp diastereomers are neither substrates nor inhibitors. We also demonstrated the catalytic activity of these Se-derivatized hammerhead ribozymes by cleaving the RNA substrate, and we found that these phosphoroselenoate ribozymes can be as active as the native. These hammerhead ribozymes mutagenized site-specifically by selenium reveal the close relationship between the catalytic activities and the replaced oxygen atoms, which provides the insight of the oxygen participation in catalysis or intramolecular interaction. This demonstrates a convenient strategy for mechanistic study of functional RNAs. In addition, the active ribozymes derivatized sitespecifically by selenium will allow convenient MAD phasing in X-ray crystal structure study.Keywords selenium derivatization; phosphoroselenoate; ribozyme; X-ray crystallography Functional RNAs play important roles in biological systems, including rRNA processing, mRNA editing, gene regulation, and RNA cleavage catalysis (1-4). X-ray crystallography is a powerful method for 3D structural and functional studies of large RNA molecules (5-7). In addition to crystallization (8,9), however, heavy atom derivatization for phase determination is still the major problem in novel structure determination of nucleic acid molecules, especially RNAs, by X-ray crystallography (6-8). On the other hand, the selenomethionine strategy was developed in order to derivatize proteins and solve the phasing problem via multi-wavelength anomalous dispersion (MAD). Recently over two thirds of novel protein structures have been determined via the selenomethionine strategy (10,11), which has clearly revolutionized protein X-ray crystallography. As selenium, sulfur, and oxygen are in the same group (Family VI) in the Periodic Table, we are attempting to develop the selenium derivatization of nucleic acids by replacing oxygen with selenium for nucleic acid X-ray crystallography, similarly to the selenium derivatization of proteins by replacing sulfur with selenium (10,11).CORRESPONDING AUTHOR FOOTNOTE: telephone: (404) 651−2915, fax: (404) 651−1416 Huang@gsu.edu. SUPPORTING INFORMATION AVAILABLE Synthesis and purification of NTPαSe analogs; Figure S1, catalysis of the modified and native hammerhead ribozymes using Mn 2+ as the metal cation. This material is available free of charge via the Internet at http://pubs.acs.org. For this goal, our laboratory is in the process of developing selenium derivatization of nucleic acids for MAD phasing (12-18), and this novel derivatization methodology has been demonstrated i...
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