Tomohisa Osaki

Synthesis and conformation of a novel bridged nucleoside with S-type sugar puckering, trans-3′,4′-BNA monomer

Abstract A novel bridged nucleoside bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton, trans-3′,4′-BNA monomer, was successfully synthesized. A 1H NMR experiment and an X-ray crystallographic analysis revealed that the sugar puckering of the 3′,4′-BNA monomer was restricted to an S-type (C3′-exo) conformation.

Acid-mediated cleavage of oligonucleotide P3' --> N5' phosphoramidates triggered by sequence-specific triplex formation.

The P-N bond in oligonucleotide P3′ → N5′ phosphoramidates (5′ -amino-DNA) is known to be chemoselectively cleaved under mild acidic conditions. We prepared homopyrimidine oligonucleotides containing 5′ -amino-5′ -deoxythymidine (5′ -amino-DNA thymine monomer) or its conformationally locked congener, 5′ -amino-2′,4′ -BNA thymine monomer, at midpoint of the sequence. The effect of triplex formation with homopurineohomopyrimidine dsDNA targets on acid-mediated hydrolysis of the P3′ → N5′ phosphoramidate linkage was evaluated. Very interestingly, it was found that the triplex formation significantly accelerates the P-N bond cleavage.

Double-stranded DNA-templated cleavage of oligonucleotides containing a P3′→N5′ linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity

We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5′-amino-2′,4′-BNA were used as a DNA-detecting probe. This modification introduced a P3′→N5′ linkage (P–N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P–N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P–N linkage should change the reactivity by altering the microenvironment. We synthesized 5′-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5′-amino-2′,4′-BNA to investigate the effect of 5′-elongation. In addition, 2′,4′-BNA/LNA or 2′,5′-linked DNA were introduced at the 3′- and/or 5′-neighboring residues of 5′-amino-2′,4′-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P–N linkage greatly affected their reaction properties. Notably, 2′,5′-linked DNA at the 3′ position flanking 5′-amino-2′,4′-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool.

Synthesis of Novel 2′-Deoxy Type Trans-3′,4′-Bridged Nucleic Acid

We newly designed and synthesized a 2 ′-deoxy type trans-3′,4′-bridged nucleic acid (trans-3′,4′-BNA) analogues bearing a 4,7-dioxabicyclo[4.3.0]nonane structure. The synthesis of the trans-3′,4′-BNA was carried out successfully from thymidine over 21 steps. The structure of trans-3′,4′-BNA was confirmed by x-ray crystallographic analysis, indicating that the furanose ring has a typical S-type conformation with C3′-exo puckering.

Cleavage of Oligonucleotides Containing a P3’→N5’ Phosphoramidate Linkage Mediated by Single-Stranded Oligonucleotide Templates

Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3’→N5’ phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.