Internal Derivatization of Oligonucleotides with Selenium for X-ray Crystallography Using MAD

Du, Quan; Carrasco, Nicolas; Teplova, M.; Wilds, Christopher J.; Egli, Martin; Huang, Zhen

doi:10.1021/ja0171097

Cited by 99 publications

(81 citation statements)

References 19 publications

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“…The product in complex with fully active A27G-substituted RNA gave the best crystals, and therefore initial efforts focused on determination of this structure. Because suitable heavy atom derivatives could not be identified using either soaking or halogen substitution approaches, we decided to rely entirely on the selenium (Se)-modification approach 23,24 , which has been successfully applied for structure solution of a DNA duplex 25 . Owing to the rapid radiation-induced decay of the crystals, six 2′-Se-methyl uridine (U Se ) and cytidine (C Se ) substitutions 24 were required for determination of the structure at a resolution of 3.0 Å by SAD (see Methods and Table 1).…”

Section: Structure Determinationmentioning

confidence: 99%

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

Serganov

Keiper

Malinina

et al. 2005

Nat Struct Mol Biol

190

225

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The majority of structural efforts addressing RNA's catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme-and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.The discovery of the catalytic activity of RNA 1,2 and the hypothesis of a prebiotic 'RNA world' 3 have expanded the scope of enzymology to include other biopolymers than proteins. The currently known natural ribozymes catalyze only a narrow range of chemical reactions, namely the hydrolysis and transesterification of internucleotide bonds 4,5 , and probably peptide bond formation 6 . However, in vitro selection and evolution have demonstrated that ribozymes are capable of accelerating a much broader reaction spectrum 7 . This finding and Correspondence should be addressed to A.J. (jaeschke@uni-hd.de) or D.J.P. (pateld@mskcc.org). COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Note: Supplementary information is available on the Nature Structural & Molecular Biology website. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript recent discoveries of metabolite-controlled RNA switches and ribozymes 8,9 suggest that RNA might have performed an even broader range of activities in the preprotein realm, and that in vitro-selected ribozymes could be analogs of the missing links in the transition from an RNA world to modern protein-dominated life 10 . Whereas high-resolution structures and biochemical investigations of several natural ribozymes provide a basic understanding of how RNA carries out phosphodiester chemistry 5,11 , little is known about how RNA catalyzes other reactions. To obtain a comprehensive picture of the catalytic abilities and limitations of ribozymes, it is thus important to expand structural and mechanistic investigations to in vitro-selected ribozymes [12][13][14] . Such structural information can be especially valuable in the determination of the minimal RNA folds required for catalysis and, therefore, could be helpful in the investigation of the origin and evolution of natural ribozymes 15 .Two examples describe the in vitro selection of ribozymes that accelerate the formation of ...

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Section: Structure Determinationmentioning

confidence: 99%

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

Serganov

Keiper

Malinina

et al. 2005

Nat Struct Mol Biol

190

225

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“…[12][13][14][15][16][17] The use of selenomethionyl proteins for multiwavelength anomalous-dispersion (MAD) phasing has revolutionized the field of protein X-ray crystallography. [18,19] This derivatization of proteins with Se has also been applied successfully in RNA structure determination through indirect derivatization of RNA that binds to the Se-derivatized protein.…”

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confidence: 99%

Efficient Enzymatic Synthesis of Phosphoroselenoate RNA by Using Adenosine 5′‐(α‐P‐Seleno)triphosphate

et al. 2005

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The discovery that RNA has a variety of biological functions in living organisms [1][2][3] has prompted the development of new methods to elucidate its structure and mechanism at the atomic level. X-ray crystallography is the method of choice for the elucidation of the three-dimensional structure of RNA macromolecules. [4,5] New developments in RNA X-ray crystallography have occurred due to advances in synchrotron radiation, diffraction data collection, [6,7] solid-phase synthesis of RNA oligonucleotides, [8] RNA crystallization, [9,10] and heavy-atom derivatization.[11] To further advance the field of RNA structure and function research, we have been working on the development of selenium derivatization for nucleic acid biochemistry and structure studies. [12][13][14][15][16][17] The use of selenomethionyl proteins for multiwavelength anomalous-dispersion (MAD) phasing has revolutionized the field of protein X-ray crystallography. [18,19] This derivatization of proteins with Se has also been applied successfully in RNA structure determination through indirect derivatization of RNA that binds to the Se-derivatized protein.[11] Motivated by the phosphoroselenoate oligonucleotide structure and function studies with MAD phasing performed by Egli and co-workers, [20] we are developing the enzymatic synthesis of phosphoroselenoate nucleic acids for X-ray crystal structure studies. We report herein the first enzymatic synthesis of phosphoroselenoate RNA (Scheme 1), containing a selenium atom that replaces one of the nonbridging oxygen atoms on the phosphate group, by in vitro transcription with T7 RNA polymerase and adenosine 5'-(a-P-seleno)triphosphate (ATPaSe).For this enzymatic synthesis, we first synthesized and characterized both diastereomeric monomers of adenosine triphosphate harboring the selenium functionality at the a-phosphate group (ATPaSe, Scheme 1) by using a modification of the procedures for the synthesis of nucleotide 5'-(a-Pthio)triphosphates (NTPaS) [21] and thymidine 5'-(a-P-seleno)triphosphate (TTPaSe).[17] The fast-and slow-moving ATPaSe isomers as represented by peaks on the reversedphase (RP) HPLC profile were termed ATPaSe I and ATPaSe II, respectively. A DNA template (55 nucleotides) was designed to allow the incorporation of 12 A residues (Figure 1 a). The generated RNA transcript (35 nucleotides) was body-labeled by using a-[32 P]-cytidine 5'-triphosphate (a-[ 32 P]CTP) in the enzymatic reaction mixture for gel electrophoresis and autoradiography. We first tested the incorporation of ATPaSe I and ATPaSe II. The results (Figure 1 b) indicated that ATPaSe I was incorporated into the RNA transcript as well as natural ATP; however, no full-length product was detected when ATPaSe II was used. Although the formation of short abortive fragments in in vitro transcription is normal, [22] surprisingly, ATPaSe I, which generated almost no short abortive sequences, led to a much cleaner reaction than natural ATP. A mixture of ATP and ATPaSe I also reduced the formation of nondesired short abortive sequ...

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“…Inspired by the natural system, our laboratory pioneered the atom-specific replacement of nucleotide O with Se ( Fig. 2) in order to obtain new insights into nucleic acid structures, and biological functions and mechanisms [38] [39]. It is exciting to explore chemical, physiological, biochemical, and structural properties of the Se-derivatized DNAs and RNAs, which also leads to a new paradigm for nucleic acids.…”

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confidence: 99%

“…Chemical synthesis of Se-derivatized nucleic acid is achieved via the solid-phase synthesis strategy by using the phosphoramidite building blocks [38] [39]. Prior to the synthesis of Se-labeled nucleic acids, there are many hurdles to overcome, including protecting and deprotecting group strategies for the Se functionalities, solubilization of the free nucleosides, the compatibility of the Se functionalities on solid-phase synthesis, and the stability of Se-derivatized nucleic acids.…”

mentioning

confidence: 99%

“…For instance, Huang and co-workers, in their effort to incorporate Se at the 5'-and 2'-positions of the nucleosides [38] [39], discovered that the selenol is unstable, as it will be rapidly oxidized to the diselenide. Sodium methyl selenide, formed by reducing dimethyl diselenide with NaBH 4 , is used as a nucleophile, and affords the methyl protection, yielding the methylseleno nucleosides [38] [39]. Initially, the introduction of Se at the 5'-position of nucleosides [38] faced some problems such as solubility of Se reagents, choice of reagents (Na 2 Se, Na 2 Se 2 , or others), and the formation of undesirable dialkyl products.…”

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confidence: 99%

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Biochemistry of Selenium‐Derivatized Naturally Occurring and Unnatural Nucleic Acids

Caton-Williams

Huang

2008

Chemistry & Biodiversity

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Selenium (Se) can provide unique biochemical and biological functions, and properties to macromolecules, including protein and RNA. Although Se has not yet been found in DNA, identification of the presence of Se in natural tRNAs has led to discovery of the naturally occurring 2-selenouridine and 5-[(methylamino)methyl]-2-selenouridine (mnm 5 se 2 U). The Se-atoms at C(2) of the modified uridines are introduced by 2-selenouridine synthase via displacement of the S-atoms in the corresponding 2-thiouridine nucleotides of the tRNAs, and selenophosphate is used as the Se donor. The research indicated that mnm 5 se 2 U is located at the first or wobble position of the anticodons in several bacterial tRNAs, including tRNA Lys , tRNA Glu , and tRNA Gln . The 2-seleno functionality on this modified nucleotide probably improves the translation accuracy and/or efficiency. These observations in vivo suggest that the presence of Se can provide natural RNAs with useful properties to better function and survival. To further investigate the biochemical and structural properties of Se-derivatized nucleic acids (SeNA), we have pioneered chemical and enzymatic synthesis of Se-derivatized nucleic acids, and introduced Se into both RNA and DNA at a variety of positions by atom-specific replacement of oxygen. This review outlines the recent advancements in chemical and biochemical syntheses, and studies of SeNAs, and their potential applications in structural and functional investigation of nucleic acids and their protein complexes.

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Internal Derivatization of Oligonucleotides with Selenium for X-ray Crystallography Using MAD

Cited by 99 publications

References 19 publications

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

Efficient Enzymatic Synthesis of Phosphoroselenoate RNA by Using Adenosine 5′‐(α‐P‐Seleno)triphosphate

Biochemistry of Selenium‐Derivatized Naturally Occurring and Unnatural Nucleic Acids

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