ABSTRACT
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. In this study, the 2'-O-methoxy derivative of Adap (2'-OMeAdap) was designed as a selective molecule for 8-oxo-rG in RNA. 8-Oxo-rG in the homopurine RNA was selectively recognized by the ODN probe incorporating Adap. In contrast, although it was not possible by the Adap-containing ODN prove due to the instability of the corresponding duplex, 8-oxo-rG in homopyrimidine RNA was selectively detected by the 2'-OMeRNA probe incorporating 2'-OMeAdap.
Subject(s)
Adenosine/chemistry , Aza Compounds/chemistry , DNA/chemistry , Fluorescent Dyes/chemistry , Guanosine/analogs & derivatives , Molecular Probes/chemistry , Oxazines/chemistry , RNA/chemistry , Adenosine/analysis , Aza Compounds/analysis , DNA/analysis , Fluorescent Dyes/analysis , Guanosine/analysis , Guanosine/chemistry , Molecular Probes/analysis , Oxazines/analysisABSTRACT
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. In this study, a new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap) was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG. The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluorescence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of 2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.
Subject(s)
2-Aminopurine/analogs & derivatives , Nucleosides/chemistry , Oxazines/chemistry , 2-Aminopurine/chemistry , Humans , Molecular StructureABSTRACT
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. After purification using HPLC, they were identified by MALDI-TOF mass measurement. The base-pairing properties showed that 8-oxo-G forms base pairs not only with rC or dC in anti-conformation, but also with rA in syn conformation within the RNA duplexes or RNA/DNA heteroduplexes.
Subject(s)
Guanosine/analogs & derivatives , Oligoribonucleotides/chemistry , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/isolation & purification , Guanosine/chemistryABSTRACT
6-O-7-N-Bis(diphenylcarbamoyl)-2-N-phenoxyacetyl-5'-O-dimethoxytrityl-2'-O-{[(triisopropyl- silyl)oxy]methyl}-8-oxoguanosine-3'-yl-ß-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. ORNs 1 and 4 containing 8-oxo-G can form base pairs with rC or dC in an anti conformation, while it can also interact with rA or dA in a syn conformation in the RNA duplex or RNA/DNA heteroduplex. The described synthetic method is therefore a useful procedure for the synthesis of ORN containing 8-oxo-G and contributes to the study of 8-oxo-G in RNA.
Subject(s)
Guanosine/analogs & derivatives , Oligoribonucleotides/chemical synthesis , RNA/chemical synthesis , Base Pairing , Guanosine/chemical synthesis , Nucleic Acid Conformation , Oligoribonucleotides/chemistry , RNA/chemistryABSTRACT
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. It was expected in this system that fluorescence of the duplex probe is first quenched by FRET, but the target DNA strand containing 8-oxo-dG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair exhibited a complete discrimination between 8-oxo-dG and dG in DNA by fluorescence enhancement.
Subject(s)
Adenosine/chemistry , DNA/chemistry , Deoxyguanosine/analogs & derivatives , Oligonucleotides/chemistry , 8-Hydroxy-2'-Deoxyguanosine , Base Sequence , Deoxyguanosine/analysis , Deoxyguanosine/chemistry , Drug Design , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/pharmacology , Hydrogen-Ion Concentration , Models, Chemical , Molecular Sequence Data , Nucleosides/chemistry , RNA/chemistry , Temperature , Time FactorsABSTRACT
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. The sp(3) oxygen atom of the carbamate unit was revealed to play a significant role in the hydrogen bonding interactions, and the pyrene group produced higher stability with 8-oxoG compared with the original 8-oxoG-clamp.
Subject(s)
Fluorescent Dyes/chemical synthesis , Guanosine/analogs & derivatives , Binding Sites , Biomarkers , Guanosine/chemical synthesis , Hydrogen Bonding , Oxidative Stress , PyrenesABSTRACT
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.
