Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope.
We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.
Tsuji
A
A
Laboratory of Molecular Biophotonics, Hamakita 434-8555, Japan. atsuji@lmbp.co.jp
Koshimoto
H
H
Sato
Y
Y
Hirano
M
M
Sei-Iida
Y
Y
Kondo
S
S
Ishibashi
K
K
D000818:Animals; D001483:Base Sequence; D001896:Boron Compounds; D019556:COS Cells; D005456:Fluorescent Dyes; D016762:Genes, fos; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic; D014162:Transfection
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger