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. 2014 Jul;42(13):8808-15.
doi: 10.1093/nar/gku538. Epub 2014 Jun 23.

Remarkable acceleration of a DNA/RNA inter-strand functionality transfer reaction to modify a cytosine residue: the proximity effect via complexation with a metal cation

Daichi Jitsuzaki  1 , Kazumitsu Onizuka  1 , Atsushi Nishimoto  1 , Ikuya Oshiro  1 , Yosuke Taniguchi  1 , Shigeki Sasaki  2
Affiliations

Remarkable acceleration of a DNA/RNA inter-strand functionality transfer reaction to modify a cytosine residue: the proximity effect via complexation with a metal cation

Daichi Jitsuzaki et al. Nucleic Acids Res. 2014 Jul.

Abstract

Modified nucleosides in natural RNA molecules are essential for their functions. Non-natural nucleoside analogues have been introduced into RNA to manipulate its structure and function. We have recently developed a new strategy for the in situ modification of RNA based on the functionality transfer reaction between an oligodeoxynucleotide probe and an RNA substrate. 2'-Deoxy-6-thioguanosine (6-thio-dG) was used as the platform to anchor the transfer group. In this study, a pyridinyl vinyl ketone moiety was newly designed as the transfer group with the expectation that a metal cation would form a chelate complex with the pyridinyl-2-keto group. It was demonstrated that the (E)-pyridinyl vinyl keto group was efficiently and specifically transferred to the 4-amino group of the opposing cytosine in RNA in the presence of NiCl2 with more than 200-fold accelerated rate compared with the previous system with the use of the diketo transfer group. Detailed mechanistic studies suggested that NiCl2 forms a bridging complex between the pyridinyl keto moiety and the N7 of the purine residue neighboring the cytosine residue of the RNA substrate to bring the groups in close proximity.

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Figures

Figure 1.
Figure 1.
(A) Conceptual illustration of the inter-strand functionality transfer from the ODN probe to the RNA substrate within the hybridized complex. (B) The 4-NH2 group of cytidine in RNA participates in a Michael addition to the vinyl group of the 2-vinylidene-1,3-diketo moiety, and the following elimination of 6-thio-dG accomplishes an S to N functionality transfer. (C) Design of the pyridinyl vinyl keto group as a new transfer group in anticipation of activation through the formation of a metal chelation complex.
Figure 2.
Figure 2.
(A) Preparation of (E)- and (Z)-FT-ODN1. (B) High pressure (or high performance) liquid chromatography (HPLC) trace of the reaction indicating 55% transfer yield of rC-modified RNA1.
Figure 3.
Figure 3.
Comparison of the transfer yields for RNA1. (A) The time course of the transfer yields. Closed squares: (E)-FT-ODN1 in the presence of NiCl2; open circles: (Z)-FT-ODN1 in the presence of NiCl2; closed diamonds: (E)-FT-ODN1 in the absence of NiCl2. (B) The transfer yields at 10 min. X represents the nucleotide opposite to the functionalized 6-thio-dG of FT-ODN1. a1 mM of Ethylenediaminetetraacetic acid (EDTA) was added. The reaction was performed at 37°C using 5 μM of RNA1 and 7.5 μM of FT-ODN1 in a buffer containing 50 mM HEPES (2-(4-(2-hydroxyethyl)piperazin-1-yl)ethane-1-sulfonic acid) and 100 mM NaCl at pH 7 in the presence or absence of NiCl2 (5 μM).
Figure 4.
Figure 4.
Effect of metal cations on the transfer yield. (A) Comparison of the yields in the presence of 5 μM of MCl2. (B) Effect of the concentration of NiCl2. The transfer yields at 1 h are compared. The reaction conditions are the same as described in the footnote to Figure 3 except that the yield was measured after 60 min.
Figure 5.
Figure 5.
Kinetic parameters at different NiCl2 concentrations. The error bars represent the standard deviation from the mean of the data obtained at different temperature. Experimental details and data are described in Supporting Information.
Figure 6.
Figure 6.
(A) A hypothetical complex of Ni2+ with the pyridinylketo unit and N7 of an adenine residue at the 5′ side. (B) Molecular modeling of the complex in the ODN1/RNA1 duplex.
Figure 7.
Figure 7.
(A) Effect of flanking base pairs on the transfer yield. M5 and M3 of FT-ODN2 are complementary bases corresponding to N3 and N5 of RNA2, respectively. (B) 7-Deaza-rG (deG) was present in RNA2. The reaction conditions are the same as described in the footnote to Figure 3 except that the yield was measured after 60 min.
Scheme 1.
Scheme 1.
The functionality transfer reaction from FT-ODN1 to RNA1 (rC).
Scheme 2.
Scheme 2.
Model experiments to determine (E)- and (Z)-stereochemistry of the transfer unit of 6-thio-dG.
Scheme 3.
Scheme 3.
1,4-Michael addition and following β-elimination leading to S-to-N transfer reaction.
Chart 1.
Chart 1.
The precursors used for attaching pyridinyl keto transfer groups to 6-thio-2′-deoxyguanosine in ODN probe.
Chart 2.
Chart 2.
Sequences of ODNs and RNAs used in this study.
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