title: Affinity labeling of a single guanine bulge.
abstract: We have developed a conceptually new method for the selective labeling of duplex DNA containing a guanine bulge with a masked form of fluorescent 2-amino-1,8-naphthyridine. A naphthyridine derivative 2 tethering DNA-alkylating epoxide was synthesized from (S)-epichlorohydrin and naphthyridine derivative 1, which selectively binds to the guanine bulge duplex. HPLC analysis of the labeling reaction of bulge duplex d(GTT GTGTTG GA)/d(CAA CA A ACC T) (TGT/A_A) with 2 showed a formation of 2-TGT adduct for the guanine bulge. The reaction proceeded for the guanine bulge and a reduced efficiency for the cytosine bulge, but not at all for adenine and thymine bulges. The site of covalent bond formation in 2-TGT was unambiguously identified at the guanine two bases away from the bulge by the use of MALDI-TOF MS analysis of the oligomer fragments produced by strand scission. The labeling reaction was also observed for the guanine bulge flanking two G-C base pairs (CGC/G_G), producing a 2:1 adduct (2.2-CGC). Upon hydrolysis of 2-TGT and 2.2-CGC with concentrated hydrogen chloride, a release of fluorescent 2-aminonaphthyridine from the adduct was clearly detected, verifying a concept of an affinity labeling of the guanine bulge with a masked fluorescent chromophore. The affinity labeling targeting of the guanine bulge is a conceptually novel method for the postsynthetic labeling of DNA. Hybridization, to the target sequence, of a probe DNA possessing one extra guanine especially between two cytosines provides a unique site for the labeling by masked fluorophore 2. The technique may have broad application in genetic typing without using a conventional synthesis of fluorescent-labeled DNA oligomers.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606-8501, Japan. nakatani@sbchem.kyoto-u.ac.jp
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nakatani
authors.forename: Souta
authors.initials: S
authors.lastname: Horie
authors.forename: Isao
authors.initials: I
authors.lastname: Saito
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes; D006147:Guanine / Q000737:chemistry*; D009287:Naphthyridines / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D013050:Spectrometry, Fluorescence
chemical_list: C427025:2-amino-1,8-naphthyridine; D000345:Affinity Labels; D015342:DNA Probes; D009287:Naphthyridines; D006147:Guanine; D004247:DNA