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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.

Jitsuzaki D, Onizuka K, Nishimoto A, Oshiro I, Taniguchi Y, Sasaki S - Nucleic Acids Res. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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(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.
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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.

Mentions: Recently, we developed a functionality transfer reaction for the site-specific modification of RNA using an ODN (oligodeoxynucleotide) probe incorporating S6-functionalized-6-thio-2′-deoxyguanosine (1) (Figure 1A). The 2-vinyliden-1,3-diketo moiety was first determined to be a transfer group, and the transfer was accomplished by a sequential reaction of a Michael addition by the 4-amino group of the cytosine base followed by β-elimination of 6-thio-dG (Figure 1B) (28). Subsequently, it was found that the transfer reaction was enhanced with selectively for the 2-amino group of the guanine base at alkaline pH or in the presence of NiCl2 at neutral pH (29,30). This method was applied to the site-specific labeling of RNA (31) and O6-methyl guanosine-containing DNA (32). The functionality transfer ODN probe (FT-ODN) is advantageous for RNA modification in that the driving force to initiate the transfer reaction is duplex formation with the target RNA. This feature represents a contrast to other methods that use activation stimuli such as photo-irradiation (21) or oxidation (33). However, it was observed that the 2-vinyliden-1,3-diketo moiety is not sufficiently stable or reactive as a transfer group for the modification of the cytosine residue. In this study, to meet the conflicting requirements of stability and reactivity, a pyridinyl vinyl ketone moiety (Pyk) was designed as a new transfer group with the anticipation that activation would occur through complexation with a metal cation, as illustrated by 3. This activation mechanism resembles the activation of DNA alkylating agents that have a quinolin-8(5H)-one substructure (34–36). This FT-ODN functionalized with an S6-vinyl pyridinyl ketone moiety showed higher stability in a buffer solution and also demonstrated, in the presence of NiCl2, a remarkably efficient transfer reaction with high selectivity for the 4-amino group of the opposing cytidine in RNA with more than 200-fold accelerated rate. A detailed mechanistic study revealed that the transfer reaction occurred specifically with (E)-geometry of the thiovinyl moiety and that NiCl2 effectively brings the two reactants in close proximity by forming a bridging complex between the FT-ODN and N7 of the purine residue neighboring the cytidine of the target RNA. In this manuscript, we describe in detail the design, synthesis and evaluation of the transfer reaction, as well as a mechanistic study on the effect of metal complexation.


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.

Jitsuzaki D, Onizuka K, Nishimoto A, Oshiro I, Taniguchi Y, Sasaki S - Nucleic Acids Res. (2014)

(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.
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Related In: Results  -  Collection

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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.
Mentions: Recently, we developed a functionality transfer reaction for the site-specific modification of RNA using an ODN (oligodeoxynucleotide) probe incorporating S6-functionalized-6-thio-2′-deoxyguanosine (1) (Figure 1A). The 2-vinyliden-1,3-diketo moiety was first determined to be a transfer group, and the transfer was accomplished by a sequential reaction of a Michael addition by the 4-amino group of the cytosine base followed by β-elimination of 6-thio-dG (Figure 1B) (28). Subsequently, it was found that the transfer reaction was enhanced with selectively for the 2-amino group of the guanine base at alkaline pH or in the presence of NiCl2 at neutral pH (29,30). This method was applied to the site-specific labeling of RNA (31) and O6-methyl guanosine-containing DNA (32). The functionality transfer ODN probe (FT-ODN) is advantageous for RNA modification in that the driving force to initiate the transfer reaction is duplex formation with the target RNA. This feature represents a contrast to other methods that use activation stimuli such as photo-irradiation (21) or oxidation (33). However, it was observed that the 2-vinyliden-1,3-diketo moiety is not sufficiently stable or reactive as a transfer group for the modification of the cytosine residue. In this study, to meet the conflicting requirements of stability and reactivity, a pyridinyl vinyl ketone moiety (Pyk) was designed as a new transfer group with the anticipation that activation would occur through complexation with a metal cation, as illustrated by 3. This activation mechanism resembles the activation of DNA alkylating agents that have a quinolin-8(5H)-one substructure (34–36). This FT-ODN functionalized with an S6-vinyl pyridinyl ketone moiety showed higher stability in a buffer solution and also demonstrated, in the presence of NiCl2, a remarkably efficient transfer reaction with high selectivity for the 4-amino group of the opposing cytidine in RNA with more than 200-fold accelerated rate. A detailed mechanistic study revealed that the transfer reaction occurred specifically with (E)-geometry of the thiovinyl moiety and that NiCl2 effectively brings the two reactants in close proximity by forming a bridging complex between the FT-ODN and N7 of the purine residue neighboring the cytidine of the target RNA. In this manuscript, we describe in detail the design, synthesis and evaluation of the transfer reaction, as well as a mechanistic study on the effect of metal complexation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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