title: Hybridization-sensitive fluorescent DNA probe with self-avoidance ability.
abstract: Hybridization-sensitive fluorescent probes have an inherent disadvantage: self-dimerization of the probe prevents the fluorescence quenching prior to hybridization with the target, resulting in a high background signal. To avoid self-dimerization of probes, we focused on a base pair formed by 2'-deoxyinosine (I) and N(4)-ethyl-2'-deoxycytidine (E). I and E bases form more stable base pairs with cytosine and guanine, respectively, compared with an I/E base pair. New hybridization-sensitive fluorescent probes, IE probes, were prepared containing three unnatural nucleotides, I, E and D(514) as a doubly thiazole orange-labeled nucleotide. The IE probes had low thermostability, sufficient to avoid self-dimerization. Absorption spectra of the IE probes exhibited a hybridization-dependent shift of the absorption maximum, suggesting that excitonic interaction was working between the thiazole orange dyes in the probe. Interdye excitonic interaction of IE probes was very effective; thus, replacement of guanine and cytosine with I and E improved the ratio of fluorescence intensities after and before hybridization (I(hybrid)/I(nonhybrid)). Although a significant weakness in fluorescence intensity was observed for several IE probes after hybridization with the target sequence when both or either of the bases adjacent to D(514) is E, a dramatic recovery of the fluorescence intensity of hybrids was observed when any E adjacent to D(514) was replaced with cytosine. Improvement of the I(hybrid)/I(nonhybrid) value by incorporation of I and E helped the design of a long probe sequence for mRNA imaging.
authors.affiliation: RIKEN Advanced Science Institute, Wako, Saitama, 351-0198, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Ikeda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kubota
authors.forename: Mizue
authors.initials: M
authors.lastname: Yuki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yanagisawa
authors.forename: Shizuho
authors.initials: S
authors.lastname: Tsuruma
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Okamoto
mesh_terms: D000042:Absorption; D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D003596:Cytosine / Q000737:chemistry; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D057054:Molecular Imaging; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D003596:Cytosine