An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5′-terminal ends of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and incorporated at the 5′-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides.

Herein, we determined a high-resolution crystal structure of a B-form DNA duplex containing consecutive dinuclear metal ion-mediated base pairs, namely, 4-thiothymine–2Ag(I)–4-thiothymine (S–2Ag(I)–S), and four Ag(I) ions form a rectangular network and the distances between the Ag(I) ions are 2.8–3.2 Å, which may indicate the existence of metallophilic attractions.

A versatile conjugatable/bioreduction-responsive protecting group for phosphodiester moieties was designed, synthesized and incorporated into oligonucleotide strands. Subsequently, controlled pore glass-supported oligonucleotides were conjugated to a variety of functional molecules using a copper-catalyzed azide-alkyne cycloaddition reaction. The functionalized protecting groups were deprotected by a nitroreductase/NADH reduction system to give “naked” oligonucleotides. This method allowed the synthesis of oligonucleotide prodrugs bearing the functionalized protecting group at the desired sites and desired residues on oligodeoxyribonucleotide (ODN) backbones.Download high-res image (127KB)Download full-size image

Cell-permeable oligodeoxyribonucleotides (ODNs) bearing reduction-activated protecting groups were synthesized as oligonucleotide pro-drugs. Although these oligonucleotides were amenable to solid-phase DNA synthesis and purification, the protecting group on their phosphodiester moiety could be readily cleaved by nitroreductase and NADH. Moreover, these compounds exhibited good nuclease resistance against 3′-exonuclease and endonuclease and good stability in human serum. Fluorescein-labeled ODNs modified with reduction-activated protecting groups showed better cellular uptake compared with that of naked ODNs.

We have examined substituted benzyl protecting groups for the phosphodiester in oligodeoxyribonucleotides. Stability of the protecting groups in buffer and rates of deprotection by glutathione (GSH) were strongly influenced by benzyl ring substituents.

Recently, metal-mediated base-pairs (metallo-base-pairs) have been studied extensively with the aim of exploring novel base-pairs; their structures, physicochemical properties, and applications have been studied. This trend has become more evident after the discovery of HgII-mediated thymine–thymine (T–HgII–T) and AgI-mediated cytosine–cytosine (C–AgI–C) base-pairs. In this article, we focus on the basic science and applications of these metallo-base-pairs, which are composed of natural bases.

A photolabile protecting group, consisting of an o-nitrobenzyl group and a 3-(2′-hydroxy-3′,6′-dimethylphenyl)-2,2-dimethylpropyl moiety, was developed for phosphodiesters in oligodeoxyribonucleotides. Deprotection was triggered by photoirradiation and subsequent spontaneous cyclization to release the naked oligonucleotide.

Thiopyrimidine pairs in DNA duplexes were unexpectedly largely stabilized by complexation with two equivalents of Ag(I) ions and their binding properties were evaluated. The metal ion-binding properties of the thiopyrimidine base pairs differed significantly from those of unpaired bases.

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine–thymine (T–T) and cytosine–cytosine (C–C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine–pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil–5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine–thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).

DNA duplexes fixed in anti-parallel and parallel orientations by introducing covalent linkages have been synthesized and metal ions, Hg(II) and Ag(I), were incorporated into pyrimidine–pyrimidine base pairs.

Very specific binding of the Ag(I) ion unexpectedly stabilized DNA duplexes containing the naturally occurring cytosine–cytosine (C–C) mismatch-base pair; because the C–C pair selectively binds to the Ag(I) ion, we developed a DNA-based Ag(I) sensor that employed an oligodeoxyribonucleotide containing C–C pairs used for Ag(I) binding sites.

It was recently demonstrated spectroscopically that RNA/DNA nucleobases can bind to metal cations in aqueous solution through coordination bonds and covalent bonds. Nitrogen-15 (15N) NMR spectroscopy was employed and shown to be a powerful tool for determining the mode of metal ion binding to nitrogen atoms in RNA/DNA molecules. This review describes 15N NMR spectroscopic characteristics in accordance with the mode of metal ion binding to nitrogen atoms. The general rules for 15N chemical shift changes, which are applicable to the determination of the metal ion binding mode of N-metallated compounds, are also described.

It was recently demonstrated spectroscopically that RNA/DNA nucleobases can bind to metal cations in aqueous solution through coordination bonds and covalent bonds. Nitrogen-15 (15N) NMR spectroscopy was employed and shown to be a powerful tool for determining the mode of metal ion binding to nitrogen atoms in RNA/DNA molecules. This review describes 15N NMR spectroscopic characteristics in accordance with the mode of metal ion binding to nitrogen atoms. The general rules for 15N chemical shift changes, which are applicable to the determination of the metal ion binding mode of N-metallated compounds, are also described.

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine–thymine (T–T) and cytosine–cytosine (C–C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine–pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil–5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine–thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).

DNA duplexes fixed in anti-parallel and parallel orientations by introducing covalent linkages have been synthesized and metal ions, Hg(II) and Ag(I), were incorporated into pyrimidine–pyrimidine base pairs.

Very specific binding of the Ag(I) ion unexpectedly stabilized DNA duplexes containing the naturally occurring cytosine–cytosine (C–C) mismatch-base pair; because the C–C pair selectively binds to the Ag(I) ion, we developed a DNA-based Ag(I) sensor that employed an oligodeoxyribonucleotide containing C–C pairs used for Ag(I) binding sites.

Thiopyrimidine pairs in DNA duplexes were unexpectedly largely stabilized by complexation with two equivalents of Ag(I) ions and their binding properties were evaluated. The metal ion-binding properties of the thiopyrimidine base pairs differed significantly from those of unpaired bases.