Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

Carrasco, Nicolas

doi:10.1093/nar/gkh325

Cited by 56 publications

(56 citation statements)

References 33 publications

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“…The synthesis of target phosphoramidite 9 and oligonucleotides is shown in Scheme 11. was activated by a 1H-triazol-1-yl group, followed by the substitution of the Se functionality, which was generated by the reduction of bis(2-cyanoethyl) diselenide with NaBH 4 [90], to generate the key intermediate 10. Then, the first Se-nucleobase phosphoramidite 9 was synthesized and incorporated into oligonucleotides by the standard solid-phase synthesis [58].…”

Section: 22mentioning

confidence: 99%

“…Encouraged by the successful structure study of the 2'-Se-DNA, a modified synthetic route was developed [58] to introduce the Se-atom efficiently into the 2'-positions of uridine (Scheme 3), which allows synthesis of the 2'-Se-U or 2'-Se-dU phosphoramidite in large quantity. The 2'-Se-cytidine building block (2'-Se-C or 2'-SedC phosphoramidite) was synthesized by the conversion of uridine to cytidine.…”

mentioning

confidence: 99%

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Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

Sheng

Huang

2010

Chemistry & Biodiversity

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It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se-Met) strategy). This selenium derivatization strategy via MAD (multi-wavelength anomalous dispersion) phasing has revolutionized protein X-ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X-ray crystal-structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O-atom at the positions 2', 4', 5', and in nucleobases and non-bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid-phase chemical synthesis and enzymatic methods, and the Se-derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein-nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2'-position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom-specific substitution of the nucleotide O-atom, better stability under X-ray radiation, and structure isomorphism. Therefore, our Se-derivatization strategy has great potentials to provide rational solutions for both phase determination and high-quality crystal growth in nucleic-acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site-specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se-derivatization strategy in nucleic acid and protein research are also described in this review.

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Section: 22mentioning

confidence: 99%

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confidence: 99%

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

Sheng

Huang

2010

Chemistry & Biodiversity

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“…29-methylseleno (29-SeCH 3 ) labeling of nucleic acids has been developed as an alternative to two broadly used phasing techniques, to the halogenation of pyrimidines and to soaking crystals in heavy atom salt solutions Teplova et al 2002;Carrasco et al 2004;Jiang et al 2007;Pallan and Egli 2007). The 29-SeCH 3 -labeling method, suited for the determination of RNA structures, relies on the crystallization of RNA molecules that have natural nucleoside(s) substituted for their 29-SeCH 3 -modified counterparts during chemical RNA synthesis.…”

Section: Introductionmentioning

confidence: 99%

A fast selenium derivatization strategy for crystallization and phasing of RNA structures

Oliéric¹,

Rieder²,

Lang³

et al. 2009

RNA

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Site-specific 29-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 29-OCH 3 -modified RNA sequences, prior to the replacement of 29-OCH 3 groups with their 29-SeCH 3 counterparts. The method exploits the similar crystallization properties of 29-OCH 3 -and 29-SeCH 3 -modified RNAs and has been successfully validated for two test cases. In addition, our data show that 29-SeCH 3 -modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.

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“…In addition to these problems, RNA molecules are prone to misfolding and adopting alternative conformations [11][12][13][14][15]. Moreover, once crystals are obtained, phasing for RNA crystals remains time-consuming and challenging compared with selenium-assisted phasing of protein crystals [11,16,17]. These problems have led RNA researchers to develop creative approaches to circumvent or ameliorate some of the problems.…”

Section: The Problem Of Rna Crystallizationmentioning

confidence: 99%

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

Piccirilli

Koldobskaya

2011

Phil. Trans. R. Soc. B

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All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3 0 ,5 0 -phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA -protein world.

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Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

Cited by 56 publications

References 33 publications

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

A fast selenium derivatization strategy for crystallization and phasing of RNA structures

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

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