Kunihiko Morihiro

Selenomethylene locked nucleic acid enables reversible hybridization in response to redox changes

Locking up selenium: A new conformationally restricted nucleic acid with a 2′,4′-selenomethylene bridge (SeLNA) can be reversibly converted into its oxidized form (SeOLNA), and the hybridization of a modified oligonucleotide was shown to be dependent on the oxidation state. A SeLNA-modified molecular-beacon-type probe (see scheme; F=fluorophore, Q=quencher) can be used as a sensor for changes in the redox environment.

Consecutive incorporation of functionalized nucleotides with amphiphilic side chains by novel KOD polymerase mutant.

Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.

Light-triggered strand exchange reaction using the change in the hydrogen bonding pattern of a nucleobase analogue

A light-triggered strand exchange reaction was developed using the change in the hydrogen-donor–acceptor pattern of a nucleobase analogue. We demonstrated that a new light-responsive nucleobase analogue derived from 4-hydroxy-2-mercaptobenzimidazole (SBNV) preferentially recognized guanine before photoirradiation and adenine after photoirradiation in duplexes. By using oligodeoxynucleotides modified with SBNV, a light-triggered strand exchange reaction targeting different mRNA fragment sequences was achieved. These results indicate that SBNV could be a powerful material for manipulating a nucleic acid assembly in a spatially and temporally controlled manner.

Synthesis of Light-Responsive Bridged Nucleic Acid and Changes in Affinity with Complementary ssRNA

The control of molecular properties through the use of external stimuli such as pH, temperature, or change in redox potential is an attractive area of research for the regulation of various biological phenomena. Among such stimuli, light is an ideal trigger because of its potential for spatiotemporal control of cellular chemistry. The compounds that can release an active molecule upon light irradiation are called “caged compounds”. In general, the properties of caged compounds, such as affinity for a specific molecule, can be changed only once (Figure 1 A). If another trigger could cause additional al-

Synthesis and Characterization of Benzylidene Acetal–Type Bridged Nucleic Acids (BA‐BNA)

Benzylidene acetal‐type bridged nucleic acids (BA‐BNAs) have a bridged structure that cleaves upon exposure to appropriate external stimuli, which induces changes in oligonucleotide properties. In particular, duplex stability and resistance to enzymatic digestion vary depending on the incorporation number and/or position of BA‐BNAs. This unit describes the synthesis of some types of BA‐BNA thymine nucleosides and the corresponding BA‐BNA phosphoramidites, as well as the incorporation of BA‐BNA nucleosides into oligonucleotides. Moreover, typical procedures that induce property changes in each BA‐BNA are described. 6‐Nitroveratrylidene and 2‐nitrobenzylidene acetal type BA‐BNA respond to photoirradiation and subsequent thiol treatment. Benzylidene and 4‐nitrobenzylidene acetal type BA‐BNAs respond to acids and reducing agents, respectively. Every BA‐BNA is derivatized into 4′‐C‐hydroxymethyl RNA by hydrolysis of the acetal‐bridged structure. Curr. Protoc. Nucleic Acid Chem. 58:1.31.1‐1.31.22.

Development of light-responsive BNA toward the spatiotemporal control of gene expression

We designed and synthesized light-responsive BNA (bridged nucleic acid), which has a photolabile group at the bridged structure. Light-Responsive BNA can change its structure by a two stages process: photoirradiation followed by nucleophilic treatment. In oligonucleotides containing light-responsive BNA moieties, binding affinity with complementary single-stranded RNA was lost upon photoirradiation, but could be restored by nucleophilic treatment.(1)