Synthesis of Selenium-Derivatized Nucleosides and Oligonucleotides for X-Ray Crystallography

Carrasco, Nicolas; Ginsburg, Dov; Du, Quan; Huang, Zhen

doi:10.1081/ncn-100105907

Cited by 68 publications

(62 citation statements)

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

et al. 2005

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“…In the case of a novel protein structure, the phase problem is usually solved by the selenomethionine (Se-Met) derivatization and multiwavelength anomalous dispersion (MAD) technique (Hendrickson 1991;Deacon and Ealick 1999). Inspired by the convenient Se-derivatization of proteins, since 1998, Huang's research group has pioneered and developed the selenium derivatization of nucleic acids for X-ray crystallography (Carrasco et al 2001;Caton-Williams and Huang 2008;. We found that the selenium-derivatized RNAs and DNAs are stable and that the selenium derivatizations do not cause significant perturbation Salon et al 2007Salon et al , 2010Sheng et al 2007Sheng et al , 2011Caton-Williams and Huang 2008;Olieric et al 2009;Hassan et al 2010).…”

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Facile synthesis of nucleoside 5′-(α-P-seleno)-triphosphates and phosphoroselenoate RNA transcription

Lin¹,

Caton-Williams²,

Kaur³

et al. 2011

RNA

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Phosphoroselenoate RNA (PSe-RNA) is nuclease resistant and has great potentials in X-ray crystal structure and function studies of noncoding RNAs and protein-RNA interactions. In order to conveniently synthesize PSe-RNA via transcription, we have developed a one-pot synthetic method for the nucleoside 59-(a-P-seleno)-triphosphates (NTPaSe) analogs without protecting any functionality of the ribonucleosides. The NTPaSe diastereomers have been purified, fully characterized, and incorporated into RNAs by T7 RNA polymerase. The transcribed RNAs are diastereomerically pure, and the Se-derivatized ribozymes are generally active. Furthermore, we have established an affinity purification strategy by using immobilized boronate to conveniently purify NTPaSe analogs. Though the affinity-purified NTPaSe analogs are diastereomeric mixtures, they can be directly used in transcription without a significant impact on the transcription efficiency. Moreover, we found that the PSenucleotide is stable during polyacrylamide gel purification, indicating that the PSe-RNAs can be purified straightforwardly for crystal structural and functional studies.

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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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“…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.…”

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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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Synthesis of Selenium-Derivatized Nucleosides and Oligonucleotides for X-Ray Crystallography

Cited by 68 publications

References 20 publications

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

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

Facile synthesis of nucleoside 5′-(α-P-seleno)-triphosphates and phosphoroselenoate RNA transcription

Biochemistry of Selenium‐Derivatized Naturally Occurring and Unnatural Nucleic Acids

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