Enzymatic Synthesis of Phosphoroselenoate DNA Using Thymidine 5‘-(α-P- seleno)triphosphate and DNA Polymerase for X-ray Crystallography via MAD

Carrasco, Nicolas; Huang, Zhen

doi:10.1021/ja0383221

Cited by 66 publications

(68 citation statements)

References 21 publications

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“…The digestion resistance of the Se-ribozyme before and after the substrate cleavage confirms the presence of the stable phosphoroselenoate modifications. This enzymatic digestion resistance is consistent with the resistance of DNA and RNA phosphoroselenoates (17,18), phosphorothioates (25), and boranophosphates (26).…”

Section: Resultssupporting

confidence: 80%

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Efficient Substrate Cleavage Catalyzed by Hammerhead Ribozymes Derivatized with Selenium for X-Ray Crystallography

2006

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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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Section: Resultssupporting

confidence: 80%

“…The resistance is consistent with resistance of DNA and RNA phosphoroselenoates (17,18), phosphorothioates (25), and boranophosphates (26). Clearly, this systematic and convenient modification of ribozyme with each NTPαSe I can assist the study and determination of its conserved sequences.…”

Section: Discussionsupporting

confidence: 65%

Efficient Substrate Cleavage Catalyzed by Hammerhead Ribozymes Derivatized with Selenium for X-Ray Crystallography

2006

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“…Nucleoside triphosphates, especially chemically modified ones, are normally synthesized by using protected nucleosides with free 59-hydroxyl groups (Ludwig and Eckstein 1989;Carrasco and Huang 2004;Brandt et al 2006). The protection is critical.…”

Section: Resultsmentioning

confidence: 99%

“…Since the diastereomer number increases exponentially by 2 n (n: the modification number), it is not practical to separate diastereomers generated by multiple phosphoroselenoates incorporated into DNA or RNA. To obtain diastereomerically pure PSe-nucleic acids, our research group has developed the enzymatic methods (Carrasco and Huang 2004;Carrasco et al 2005;Brandt et al 2006). In order to prepare phosphoroselenoate RNA (PSe-RNA) (Fig.…”

Section: Introductionmentioning

confidence: 99%

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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“…[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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Enzymatic Synthesis of Phosphoroselenoate DNA Using Thymidine 5‘-(α-P- seleno)triphosphate and DNA Polymerase for X-ray Crystallography via MAD

Cited by 66 publications

References 21 publications

Efficient Substrate Cleavage Catalyzed by Hammerhead Ribozymes Derivatized with Selenium for X-Ray Crystallography

Efficient Substrate Cleavage Catalyzed by Hammerhead Ribozymes Derivatized with Selenium for X-Ray Crystallography

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

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

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