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Bioconjugate Chem. 2010, 21, 1508–1512
Activation and Alteration of Base Selectivity by Metal Cations in the Functionality-Transfer Reaction for RNA Modification Kazumitsu Onizuka, Yosuke Taniguchi, and Shigeki Sasaki* Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, and CREST, Japan Science and Technology Agency, 4-1-8 Motomachi, Kawaguchi, Saitama 332-0012, Japan. Received March 9, 2010; Revised Manuscript Received June 14, 2010
Previously, we reported that the 2-methylidene-1,3-diketone unit of 6-thioguanosine transferred selectively to the amino group of cytosine at pH 7.0 and that its selectivity was changed to the guanine base at pH 9.6. In this study, it was found that the functionality-transfer reaction enhanced selectivity for the guanine base in the presence of divalent transition metal cations such as Ni2+ and Co2+ at pH 7.4.
INTRODUCTION A number of oligodeoxynucleotides (ODNs) with an attached chemically reactive accessory molecule have been developed to perform chemical modifications of DNA or RNA in a sequence-specific manner (1, 2). In particular, there is increasing demand for methods of the site-specific modifications of RNA because of potential applications as innovative biological tools (3-6) and therapeutic methods (7-9). The accessory molecule needs to be stable, and its reactivity is desired to be induced when it is needed. UV irradiation is frequently used as an exogenous activation signal (10-13). We have been developing an original strategy for the induction of the reactivity of the accessory molecules by taking advantage of minute environmental changes accompanied by nucleic acid hybridization. The first example was demonstrated by the hybridization-promoted cytosine-selective alkylation using the sulfide derivative of 2-amino-6-vinylpurine nucleoside, in which the stable sulfide precursor was spontaneously activated to the active vinyl species within the complementary duplex having the cytosine base at the target site (14-16). This strategy was then expanded to nitrosyl group transfer from the sulfur atom of 6-thioguanosine to the amino group of the cytosine base (17). Its reactivity was induced in close proximity to the cytosine base in the complementary duplex that affected the nitrosyl transfer to the amino group of the cytosine base. Cytosine deamination following the nitrosyl transfer reaction suggested its potential application as an artificial tool for RNA editing. For further expansion of this strategy, we developed a functionality-transfer reaction to the amino group of the cytosine base using the ODN incorporating S-(2-methylidene-1,3-diketone)-6-thioguanine, and the reaction enabled the site-specific covalent modification of RNAs (Figure 1A and B) (18). Interestingly, it was found that the transfer reaction was greatly accelerated toward the guanine base under alkaline conditions and led to a change in base selectivity from rC at pH 7.0 to rG at pH 9.6 (Figure 1C) (19). This functionality-transfer reaction has been applied to the site selective transfer of the fluorescent pyrene unit to rG in the RNA substrate (19). This method is characteristic in that it might be applied to the modification of large RNA and also in that base selectivity can be controlled by pH change. If the reactivity can be activated by a factor other than pH, this method would find wider utility in chemical * Corresponding author. Tel: +81-92-642-6615. Fax: +81-92-6426786. E-mail:
[email protected].
Figure 1. Concept of the functionality-transfer reaction (A) and selective reaction leading to C-modification at pH 7 (B) and G-modification at pH 9.6 (C).
modification of natural RNA. The selectivity change was reasonably explained in terms of an increase in the nucleophilicity of the 2-amino group by the enolate formation of the guanine base under alkaline conditions. These considerations led us to hypothesize that transition metal ions such as Ni2+, Co2+, Cu2+, and Zn2+, which are known to coordinate at the N7 position of the guanine base and decrease the pKa value of the N1 position (20), might enhance the reactivity of rG under neutral conditions. In this article, we report that Ni2+and Co2+ effectively enhance the reactivity of rG to switch the base selectivity to rG from rC under neutral conditions.
EXPERIMENTAL PROCEDURES General Methods. All ODNs were synthesized by using an automated DNA synthesizer (Applied Biosystems 394 DNA/ RNA synthesizer) at a 1.0 µmol scale. The structures of synthetic ODNs were confirmed by MALDI-TOF MS measurement (Bruker Daltonics Microflex instrument). MALDI-TOF MS results are summarized in Table 2S (Supporting Information). All ORNs were purchased from Gene Design Inc. or Genenet. Functionality-Transfer Reaction from the Functionalized ODN1F (X ) GS(Me,Ph)) to ORN2 in the Presence of NiCl2 as the General Procedure for the Functionality-Transfer Reaction. ODN1F (X ) GS(Me,Ph) was freshly prepared before the functionality-transfer reaction by mixing ODN1 and 3-
10.1021/bc100131j 2010 American Chemical Society Published on Web 06/29/2010
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Figure 4. Concentration dependency of the metal cation on the functionality-transfer reaction. The transfer reactions were performed by using 15 µM S-functionalized ODN1F-GS(Me, Ph) and 10 µM target ORN2 (G) in 50 mM MES buffer containing 100 mM NaCl in the presence of 0-1 mM MCl2 at pH 7.4 for 1 h, followed by HPLC with UV monitoring at 254 nm. Figure 2. Structure of S-functionalized 6-thioguanosine (A) and the sequences of the ODN probes, the target ORN and ODN (B).
chloromethylene-4-phenylbutane-2,4-dione in carbonate buffer (pH 10). The solution of ODN1F (X ) GS(Me,Ph)) and a solution of the target ORN2 were mixed in MES buffer containing NaCl and NiCl2, and the mixture was kept at 25 °C. The reaction mixture was analyzed by HPLC with UV monitoring at 254 nm or FAM fluorescence at 518 nm with emission at 494 nm. Column, SHISEIDO C18, 4.6 × 250 mm; solvent A, 0.1 M TEAA buffer; B, CH3CN; B, 10% to 30% /20 min, linear gradient; flow rate, 1.0 mL/min. UV Melting Temperature Measurements. UV melting temperatures were measured using 1.3 µM of each ODN and ORN strands in MES buffer (pH 7.4) containing 100 mM NaCl and 1 mM MCl2 with monitoring at 260 nm by a UV-Vis spectrophotometer. Circular Dichroism Measurements. CD spectra were measured by using 2.5 µM of each ODN and ORN strand in 50 mM MES buffer (pH 7.4) containing 100 mM NaCl and 1 mM MCl2 at room temperature in a cylindrical quartz cell with a path length of 0.1 cm.
RESULTS AND DISCUSSION Figure 2 summarizes the structure of the ODN1 probe incorporating the 6-thioguanosine analogue functionalized with the phenyl-1,3-diketone unit and ORN2 and ODN3 as the targets. We first investigated the effect of metal dichloride
on the functionality-transfer reaction to rG of RNA at pH 7.4 using ODN1F (X ) GS(Me,Ph)) (Figure 3). The transferred yields at an hour are compared in Figure 3A. A remarkable increase in the reaction yield was observed in the presence of a transition metal cation such as Ni2+, Co2+, Zn2+, or Mn2+. The HPLC profile after 60 min in the presence of Ni2+ is shown as an example in Figure 3B representing the formation of the new product together with the decreased ORN2 and the reproduced ODN1. The transferred structure of the new peak eluting at a retention time of 9.6 min was supported by the MALDI-TOF MS measurements (Table 2S (Supporting Information), ORN2F). The HPLC analysis of the hydrolysates of the products with bacterial alkaline phosphatase (BAP) and snake venom phosphodiesterase (SVPD) has clearly indicated that the modified rG obtained in the presence of NiCl2 at pH 7.4 is identical to that obtained at pH 9.6 (Figure 9S, Supporting Information), which has been concluded to be the 2-amino modified guanosine in the previous study (19). Alkaline earth metal ions such as Ba2+, Ca2+, and Mg2+ slightly promoted the reaction. In contrast, Cu2+ and Fe2+ did not activate the reaction (Figure 3A). As a result, it turned out that Ni2+, Co2+, Zn2+, and Mn2+ were suitable ions to activate the G transfer reaction. According to the IrvingWilliams series (21), the coordination ability of the metal cations to the nitrogen atom such as N7 of guanosine is in the order of Zn2+ < Cu2+ > Ni2+ > Co2+ > Fe2+ > Mn2+,
Figure 3. Effect of metal cation on the functionality-transfer reaction (A) and the HPLC profiles on the Ni2+-activated transfer reaction (B). The transfer reactions were performed by using 15 µM S-functionalized ODN1F-GS(Me, Ph) and 10 µM target ORN2 (G) in 50 mM MES buffer containing 100 mM NaCl in the presence of 1 mM MCl2 at pH 7.4 for 1 h, followed by HPLC with UV monitoring at 254 nm.
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Table 1. UV Melting Temperatures of the Duplexes Formed with ODN and ORN in the Presence of MCl2a Metal (M ))
Tm (°C)
∆Tm (°C)
none Ba Ca Mg Mn Co Ni
47.8 48.9 48.9 49.0 50.0 53.5 55.5
+1.1 +1.1 +1.2 +2.2 +5.7 +7.7
a The Tm values were measured using 1.3 µM S-functionalized ODN1F-GS(H, Ph) and 1.3 µM of the target ORN2 (G) in 50 mM MES buffer (pH 7.4) containing 100 mM NaCl in the presence of 1 mM MCl2.
Figure 5. CD spectra in the presence of MCl2. CD spectra were measured using 2.5 µM duplexes in 50 mM MES buffer (pH 7.4) containing 100 mM NaCl and 1 mM MCl2.
Figure 6. Effect of the metal cation on the transfer reaction rate. The reactions were performed by using 15 µM S-functionalized ODN1FGS(Me, Ph) and 10 µM target ORN2 (G) in 50 mM MES buffer containing 100 mM NaCl in the presence of 1 mM MCl2 or 0.1 mM CuCl2 at pH 7.4, followed by HPLC with UV monitoring at 254 nm.
followed by the alkaline earth metals (Mg2+, Ca2+, and Ba2+). The reaction yields in the presence of these metals followed
this order, except for the reaction with Cu2+ and Fe2+. One may assume that an excess of these cations induced unfavorable changes in RNA. In order to examine this hypothesis, the concentration dependency of Cu2+ or Fe2+ was compared with that of Ni2+, Zn2+, or Mg2+ (Figure 4). The transfer reaction was remarkably activated with Ni2+ at more than 0.05 mM, and its effect reached a plateau at concentrations higher than 0.5 mM. A similar concentration-dependent activation was observed with Zn2+. Activation by Mg2+ was small even at 1 mM. However, in the case of Cu2+ or Fe2+, the reaction yield was highest at 0.1 mM concentration. To check whether Cu2+ and Fe2+ decrease the thermal stability of the duplex, Tm values and CD spectra were investigated in the presence of the metal cation at pH 7.4 using ODN1F (X ) GS(H,Ph)) containing the nonreactive monoketone unit and the complementary ORN2 (Y ) rG). Clear melting curves were not observed in the presence of 1 mM of Cu2+, Fe2+, and Zn2+. Decomposition of the RNA strand at high temperature in the presence of Zn2+ was confirmed by HPLC analysis after Tm measurement (22). Mn2+, Co2+, and Ni2+ increased the Tm values in the range of 2-8 °C (Table 1). The CD spectra in the presence of 1 mM of Ni2+ and Zn2+ were similar to those taken in the absence of divalent metal cations, indicating that the duplex was in a conformation between the A and B types (Figure 5). However, Cu2+ decreased the intensity of the CD spectra, and the CD spectra in the presence of Fe2+ indicated a distortion of the duplex conformation. Therefore, the low reactivity in the presence of Cu2+ and Fe2+ can be attributable to the induction of unfavorable conformations in the transfer reaction (23). The effect of metal cations on the transfer reaction rate was also investigated with 1 mM of Ni2+, Co2+, Mn2+, Zn2+, and Mg2+, or 0.1 mM of Cu2+ (Figure 6). Fe2+ was not used because it significantly distorted the duplex conformation as described above. The transfer reaction rate was remarkably high with Ni2+ and Co2+, and the yield reached a plateau over 80% within 120 min. The yield of the transferred products was constantly increased even after 120 min in the presence of Mn2+ and Mg2+, although the reaction rate was lower than that with Ni2+ or Co2+. However, in the case of Zn2+ and Cu2+, the yield reached a plateau after 120 min at lower levels. Decomposition of the functional ODN probe in the presence of Zn2+ and Cu2+ was evidenced by the HPLC analysis, resulting in low yields of the transfer products. To determine whether metal coordination to N7 plays a role in activation, we next investigated the effect of the nucleobase structure. The transfer reaction proceeded with
Figure 7. Comparison of the reactivity of the guanine analogues in the presence of the metal cation. The transfer reactions were performed by using 15 µM S-functionalized ODN1F-GS(Me, Ph) and 10 µM target ODN3 (Y) in 50 mM MES buffer containing 100 mM NaCl in the presence of 1 mM MCl2 at pH 7.4 for 1 h (A) or for 0-6 h (B), followed by HPLC with UV monitoring at 254 nm.
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Figure 8. Base selectivity of the transfer reaction in the presence of divalent transition metal cations (A) and the HPLC profiles of the transfer reaction in the presence of NiCl2 (B). The transfer reactions were performed by using 1.5 µM S-functionalized ODN1-GS(Me, Ph) and 1.0 µM target ORN2 (Y) in 50 mM MES buffer containing 100 mM NaCl in the presence of 1 mM MCl2 at pH 7.4 for 1 h, followed by HPLC with FAM fluorescence monitoring at 518 nm with emission at 494 nm.
the ODN targets in a similar fashion with the ORN targets with respect to metal cation activation (Figure 10S, Supporting Information) and guanine selectivity (Figure 11S, Supporting Information), although the reaction efficiency was lower than that with RNA substrates. Therefore, the experiments were done with the ODN targets. It was expected that the reaction of 8-oxoguanine (8-oxo-dG) and 7-deazaguanine (7-deazadG) would not be accelerated because of the lack of N7 for coordination with the metal cations. The reactivities of dG, 8-oxo-dG and 7-deaza-dG in the presence or absence of the metal cations are compared in Figure 7. 7-DeazadG hardly produced the transferred products either in the presence or the absence of the metal cations. The reactivity of 8-oxo-dG was not affected by the additional metal cations (Figure 7A). In contrast, the transfer reaction to dG was affected in the presence of metal cations, especially by Ni2+ and Co2+. These results support the idea that the metal coordination to N7 is a major determinant of the rate of acceleration. All of the transfer reactions to dG, oxodG, and 7-deaza-dG were enhanced under alkaline conditions as previously reported (Figure 12S, Supporting Information) (19). Thus, it may be explained, as initially expected, that the enolate becomes favored by metal coordination on N7 of guanine to enhance the reactivity of the 2-amino group of dG under neutral conditions. The base selectivities at pH 7.4 in the presence of transition metal cations were investigated using the S-functionalized ODN1F probe and the complementary ORN2 (Y ) rG, rC, rA, rU) (Figure 8). The HPLC charts of the reaction mixture at 1 h are compared in Figure 8B. In the presence of transition metal cations, especially Ni2+ and Co2+, the reaction efficiency with rG was very high compared with that of rC, and no reaction was observed for rA or rU. These results have clearly shown that the functionality-transfer reaction is enhanced selectively for rG by the metal cations to cause selectivity change from rC to rG.
CONCLUSIONS In conclusion, it has been demonstrated that the functionality-transfer reaction to the guanine base in RNA was greatly and selectively enhanced in the presence of transition metal cations under neutral conditions, resulting in a selectivity change from rC to rG. This method enables the modification of RNA site-selectively with high efficiency under near physiological conditions and is expected to be useful for the
study of RNA such as through an application to modify large intact RNA molecules.
ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research (S) from the Japan Society for the Promotion of Science (JSPS) and CREST from the Japan Science and Technology Agency. We are grateful for the Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists (K.O.). Supporting Information Available: MALDI-TOF/MS of ODN, ORN, HPLC analysis of the hydrolysates of the modified ORN, comparison of the yield of the transfer reaction in the presence of the metal cations. This material is available free of charge via the Internet at http://pubs.acs.org.
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