Recognition and detection of 8-oxo-rG in RNA using the DNA/OMeRNA chimera probes containing fluorescent adenosine-diazaphenoxazine analog.

Recent studies indicate that oxidative damage to RNA results in dysfunction of translation and eventual pathogenesis. A representative oxidized base in RNA is 8-hydroxyguanosine (8-oxo-rG), however, unlike its DNA counterpart (8-oxo-dG), its role in pathogenesis has not attracted much attention until recently. The 2'-deoxyadenosine derivative with a diazaphenoxazine skeleton at the 6-amino group (Adap) was shown to be selective for 8-oxo-dG in DNA.

Synthesis of 8-oxoguanosine phosphoramidite and its incorporation into Oligoribonucleotides.

The detailed synthetic protocol for preparation of the phosphoramidite of an oxidatively damaged ribonucleotide, 8-oxoguanosine (8-oxo-G), and its incorporation into RNA are described. The O(6)- and N(7)-bisdiphenylcarbamoyl-protected 8-oxoguanosine phosphoramidite was synthesized as a new phosphoramidite precursor unit for the synthesis of RNA. It was successfully incorporated into the RNA sequences, and the synthesized RNAs were completely deprotected with 28% aqueous ammonia solution at 55°C for 24 hr.

2,6-diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3-diazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in DNA.

8-oxo-2'-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer. Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3-diazaphenoxazine (Adap), forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching.

Synthesis of the oligoribonucleotides incorporating 8-oxo-guanosine and evaluation of their base pairing properties.

6-O-7-N-Bis(diphenylcarbamoyl)-2-N-phenoxyacetyl-5'-O-dimethoxytrityl-2'-O-{[(triisopropyl- silyl)oxy]methyl}-8-oxoguanosine-3'-yl-β-cyanoethyl-N,N-diisopropylphosphoramidite (5) was synthesized as a new phosphoramidite precursor unit for the synthesis of RNA. Compound 5 was successfully incorporated into the middle of the RNA sequences, and the synthesized RNAs were identified by MALDI-TOF mass measurements. Their properties were evaluated for formation of the RNA duplex and RNA/DNA heteroduplex.

OFF-to-ON type fluorescent probe for the detection of 8-oxo-dG in DNA by the Adap-masked ODN probe.

We have recently reported that Adap (adenosine-1,3-diazaphenoxazine) is an artificial nucleoside analogue for the specific recognition by multiple hydrogen bonding and that its fluorescence is selectively quenched with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA. We now report the development of a new OFF-to-ON type FRET probe, in which one strand contains Adap and another contains natural nucleotides for the formation of a less stable double strand. Each strand was labeled with Cy3 or BHQ2 at the 5'-end or 3'-end, respectively.

Synthesis of new derivatives of 8-oxoG-clamp for better understanding the recognition mode and improvement of selective affinity.

8-Oxoguanosine (8-oxoG) is a representative metabolite derived by the oxidation of guanosine (G) and is regarded as a marker of oxidative stress in the cells. We previously reported the 8-oxoG-clamp as the first fluorescent probe for detection of 8-oxoG. In this study, new 8-oxoG-clamp derivatives having a variety of N-functional groups were synthesized and their recognition properties were investigated.

Development of a specific fluorescent probe for 8-oxoguanosine.

8-Oxoguanosine (8-oxoG) is derived from the oxidation of guanosine (dG) and is known to induce transversion mutations (G:C to T:A) in DNA. We have already reported the fluorescent probe "8-oxoG-clamp", which shows a selective fluorescence quenching phenomenon toward 8-oxoG. In this study, attachment of an additional fluorophore to 8-oxoG-clamp was investigated as a strategy for the detection of 8-oxoG by a fluorescence color change attributed to the 8-oxoG-clamp.