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Non-Enzymatic RNA Hydrolysis Promoted by the Combined Catalytic Activity of Buffers and Magnesium Ions Mounir G

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Non-Enzymatic RNA Hydrolysis Promoted by the Combined Catalytic Activity of Buffers and Magnesium Ions Mounir G Non-Enzymatic RNA Hydrolysis Promoted by the Combined Catalytic Activity of Buffers and Magnesium Ions Mounir G. AbouHaidar3 and Ivan G. Ivanovb* a Department of Botany, Virology Group, University of Toronto, 25 Willcocks Street, Toronto, Ontario. Canada M5S 3B2 b Institute of Molecular Biology, Department of Genetic Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Fax: (+3592) 736227: 723507. E-mail: [email protected]; [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 54c, 542-548 (1999); received January 20/March 23. 1999 RNA, RNA Degradation, RNA Hydrolysis, Acid-Base Catalysis, Magnesium Catalysis Although Mg2+ is an important cofactor for the specific degradation of RNA by ribozymes, it is not considered as a typical chemical nuclease. In this study we show that in combination with common buffers such as tris(hydroxymethyl)aminomethane and sodium borate. Mg2+ is a powerful catalyst for the degradation of RNA. pH and temperature are found to be the principal factors for the efficient degradation of RNA. Whereas in Tris-HCl/Mg2+ the effi­ cient cleavage starts at pH values higher than 7.5 and temperatures higher than 37 °C, in sodium borate RNA degradation begins at pH 7.0 and at 37 °C. RNA hydrolysis promoted under the combined catalytic activity of buffer/Mg2+ results in partially degraded RNA and negligible amounts of acid-soluble material. Reaction is insensitive to the concentration of monovalent cations but is completely prevented by chelating agents (ED TA and citrate) at concentrations exceeding that of Mg2+. Borate-magnesium reaction is inhibited also by some polyvalent alcohols (glycerol) and sugars. Introduction As a result, the necessity of divalent ions for the ribozyme reaction is commonly accepted but the The presence of a 2'-OH group in the ribose exact role of magnesium in the transesterification moiety is the main reason for chemical instability reaction accompanying the cleavage of the phos­ of RNA in comparison with DNA. Unlike DNA, phate diester bond is still disputable. It should be RNA is easily hydrolysed in aqueous solutions in mentioned that the Mg2+ catalysed hydrolysis is the presence of alkali or heavy metal ions (M or­ considered as independent of the buffer compo­ row, 1994). On the other hand, RNA as a single­ nents and it is mainly discussed in the context of stranded biopolymer, binds naturally Mg2+ and sequence specific cleavage of RNA. Therefore, other metal ions, which are necessary for the main­ Mg2+ itself is not considered as a degrading agent tenance of its higher ordered structures (Morrow, for RNA at neutral pH values (Breslow and Hu­ 1994; Van Atta and Hecht, 1994). Whereas Mg2+ ang, 1991; Morrow, 1994) but as a secondary struc­ is not a co-factor for the enzymatic hydrolysis of ture stabiliser. RNA (Morrow, 1994), Mg2+ or Mn2+ are indis­ In our laboratories we have observed an in­ pensable for the activity of catalytic RNAs (in- creased instability of RNA in certain buffers in the trons and ribozymes; for review see Dahm and presence of magnesium salts. Surprisingly, some of Uhlenbeck, 1991; Dahm et al., 1993; Van Atta and them like tris(hydroxymethyl)aminomethane hy­ Hecht, 1994). drochloride (Tris-HCl) and sodium borate, are Due to the increased interest in the mode of among the most commonly used buffers in the action of ribozymes, the role of Mg2+ and other RNA research. In this report, we provide data divalent metal ions for the specific non-enzymatic about the factors affecting RNA hydrolysis in hydrolysis of RNA has been extensively studied buffers containing Mg2+ and discuss the possible (Dahm and Uhlenbeck, 1991; Dahm et al., 1993; mechanism of that reaction. Long and Uhlenbeck. 1993; Uchimaru et al., 1993; Van Atta and Hecht, 1994). 0939-5075/99/0700-0542 $ 06.00 © 1999 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D M. G. AbouHaidar and I. G. Ivanov • Non-Enzymatic RNA Hydrolysis 543 Materials and Methods U/mg RNA) at 37 °C for 30 min and the other half was extensively extracted with phenol, phenol/ Preparation o f RNA chloroform and chloroform, precipitated with eth­ Tobacco mosaic virus (TM V) RNA was pre­ anol and dissolved in TE buffer (0.5-1.0 mg/ml). pared by phenol extraction of purified TMV. Total For 5'-end labelling, samples of 3 [il were treated RNA from E. coli was prepared according to cur­ with T4 polynucleotide kinase in the presence of rent protocols (Sambrook et al., 1989). RNA prep­ y-[32P]-ATP (2 |.iCi) in 50 jil reaction buffer at arations were extracted consecutively with phenol/ 37 °C for 1 h and for 3'-end labelling the same chloroform and chloroform. RNA was used only amount of RNA was incubated overnight with if no detectable degradation was found after an RNA ligase and [32P]-CPC (2 |iCi) in 50 \i\ at 4 °C. overnight incubation in TE buffer (10 m M Tris- Reactions were stopped by adding an equal vol­ HC1, 1 mM EDTA) at 37 °C. E. coli rRNAs and ume of ice-cold 10% TCA. After 15 min incuba­ tRNAs were either purchased from Boehringer tion on ice precipitates were collected by centrifu­ Manheim or purified from total bacterial RNA. gation at 4 °C and washed 3 times with ice-cold RNA fragments of defined length were pre­ 5% TCA. Precipitates were dissolved in 50 ill 1n pared by in vitro transcription of DNA fragments KOH and incubated for 1 h at 60 °C. Samples were cloned downstream of the T7 RNA polymerase chilled on ice and 50 j_il of ice-cold 1n H C104 were promoter in pBS M13+ (Stratagene) using T7 added. After 5 min the white precipitate (K C 104) RNA polymerase and ribonuclease inhibitors was removed by centrifugation and the water (New England BioLabs). Template DNA was di­ phase was concentrated (if necessary) in a Speed- gested by DNase (RNase free) and RNA samples Vac centrifuge to about 10,000 cpm/[il of 32P ra­ were extracted extensively with phenol, phenol/ dioactivity (counted by Cerenkov). 32P-labelled chloroform and chloroform. All RNA prepara­ terminal nucleotides were separated by electro­ tions were stored at -20 °C in TE buffer at a con­ phoresis on a Whatman #1 paper in a pyridine centration of 1 mg/ml. (0.5%) /acetic acid (10%) pH 3.5 buffer at 40 V/ cm for 30 min. The radioactive spots were visua­ Hydrolysis and electrophoresis of RNA lised by autoradiography, cut out and 32P-radioac- tivity was measured by Cerenkov in a Beckman Conditions for degradation of RNA in buffers scintillation counter. are given in the figure legends. Agarose gel (1% or 1.5% ) electrophoresis was carried out in Tris/ borate, EDTA, pH 7.5 buffer containing 0.5 [ig/ml H P L C analysis o f ribonucleotides ethidium bromide at room temperature. Values for Three di-ribonucleoside monophosphates (UpU, PH were determined for each buffer at the indi­ CpU and ApU) were hydrolysed overnight in both cated temperature. buffers 50 m M Tris-HCl, pH 8.0, 20 m M MgCl 2 and 50 m M sodium borate, pH 7.5, 20 m M MgCl 2 under Terminal nucleotide analysis the conditions described above and the degrada­ 32P-labelled RNA preparations were prepared tion products were analysed by HPLC (Waters). for 5'- or 3'-terminal nucleotide analysis as fol­ Two type of samples were used as referents for lows: TMV RNA and rRNA were partially de­ identification of the degradation products: 1) the graded in: 1) 50 mM Tris-HCl, pH 8.0,20 mM MgCl 2 same di-nucleoside monophosphates hydrolysed at 55 °C for 2 h; 2) 50 mM sodium borate, pH 7.5, in 10 m M KOH at 37 °C for 2 h, and 2) commercial 20 mM MgCl 2 at 55 °C for 2 h; 3) 10 mM KOH at nucleoside (U) and ribonucleotides (3'-UMP. 3'- 37 °C for 30 min. RNA was precipitated with 5% CMP, 3'-AMP, 2'-UMP, 2'-AMP, 2',3'-UMP, 2\3'- (final concentration) ice-cold trichloro acetic acid CMP and 2',3'-AMP). The chromatographic con­ (TCA) for 15 min and the precipitate was washed ditions for separation of reaction products were consecutively with ice-cold 90% ethanol, 70% eth­ the following: amino column (Spherisorb-NH2, anol and then dissolved in TE buffer at a concen­ 5 |il particles), flow rate 0.8 ml/min, isocratic elu­ tration of 0.5-1.0 mg/ml. One half of the RNA tion with 10 m M KH2P 0 4/acetonitrile (30/70 v/v), sample was treated with alkaline phosphatase (0.5 pH 4.6. 544 M. G. AbouHaidar and I. G. Ivanov • Non-Enzymatic RNA Hydrolysis The difference between the retention times of the nucleoside-2'-monophosphates and nucleo- side-3'-monophosphates was approximately 5 min (i.e. 17 min for 2'-UM P and 22 min for the 3'- UMP). - f « Chemicals and lahware 1 2 3 4 5 6 7 8 All reagents used in this study were of ultra pure grade practically free from metal ions and Fig. 1. Stability of TMV RNA in different buffers at pH 8.0. TMV RNA (1 |ig) was incubated at 55 °C for RNase free. Buffers were prepared with diethyl- 2 hours in distilled water (lane 8) or 50 mM buffers (lanes pyrocarbonate (DEPC) treated and autoclaved 1 -7 ) supplemented with 20 mM MgCl2 (lanes 2. 4. 6, 8) water. All reactions and manipulations were car­ or free of Mg2+ (lanes 1, 3, 5. 7). Buffers: threonine (1); ried out in DEPC pre-treated glass and plastic glycine (2, 3); sodium borate (4, 5) and Tris-HCl (6, 7).
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