title: Molecular design of Cy3 derivative for highly sensitive in-stem molecular beacon and its application to the wash-free FISH.
abstract: We herein describe a novel in-stem molecular beacon (ISMB) containing multiple Cy3-quencher pairs on d-threoninol scaffolds in the stem region. The designed Cy3 derivative was not significantly quenched by the adjacent nucleobases, self-quenching of the fluorophore was minimal, and the fluorophore did not severely destabilize the duplex. Using newly designed Cy3, we synthesized ISMBs containing two Cy3 moieties. The signal to background ratio of the ISMB containing two Cy3 moieties was above 100, whereas that with one Cy3 was 30. A Cy3-derivative containing ISMB used in a fluorescence in situ hybridization (FISH) detected endogenous β-actin mRNA in fixed cells without need for washing procedures.
authors.affiliation: Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: kashida@nubio.nagoya-u.ac.jp.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Kashida
authors.affiliation: Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Osawa
authors.affiliation: Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Morimoto
authors.affiliation: Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; EcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kamiya
authors.affiliation: Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Electronic address: asanuma@nubio.nagoya-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Asanuma
mesh_terms: D000199:Actins / Q000235:genetics; D000605:Amino Alcohols / Q000737:chemistry*; D001483:Base Sequence; D002072:Butylene Glycols / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
chemical_list: D000199:Actins; D000605:Amino Alcohols; D002072:Butylene Glycols; D002232:Carbocyanines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C089187:cyanine dye 3; C532652:threoninol
keywords: Cy3; DNA; Fluorescence in situ hybridization; Molecular beacon