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

Abstract

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...

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

“…3). Consistent with our previous report (Carrasco et al 2005;Brandt et al 2006), clearly, T7 RNA polymerase can efficiently recognize the NTPaSe I diastereomers (Sp) at the same level as the native NTPs (Fig. 3), while the NTPaSe II diastereomers (Rp) were not recognized.…”

“…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.…”

“…In our previous study, NTPaSe analogs were purified by HPLC, which is labor-intensive and has a low capacity for larger-scale synthesis (Carrasco et al 2005;Brandt et al 2006). Thus, we developed the boronate affinity method for purification of NTPaSe analogs.…”

“…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.…”

“…1) through transcription, we first synthesized the 59-(a-P-seleno)-triphosphates (NTPaSe) analogs ( Fig. 1; Carrasco et al 2005;Brandt et al 2006). Considering broad applications of the NTPaSe analogs and PSe-RNAs, it is necessary to develop a convenient strategy to synthesize the NTPaSe analogs and PSe-RNAs on a large scale.…”