Development of real time imaging of specific messenger RNA in a living cell using artificial antisense nucleic acids.
Real time imaging of mRNA in living cell is powerful tool in investigating the role of mRNA. Here we developed the method for visualizing specific mRNAs in living cell. The proposed method contains hybridization of antisense nucleic acids with mRNA transcripted inside cell. As antisense nucleic acid, 2'O-methyl RNA and Peptide Nucleic Acid (PNA) were chosen because these artificial nucleic acids are nuclease resistant and have high affinity to mRNA. Two antisense probes labelled with different fluorescent dyes were designed and prepared. When two antisense probes were hybridized to target human c-fos mRNA, two fluorescent dyes became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. Such FRET signals were detected when 2'O-methyl RNA and Peptide Nucleic Acid were used as antisense probes. The biggest signal was detected when 2'O-methyl RNA as donor probe and PNA as acceptor probe were used. The timecourse study indicated two antisense probes can hybridize fast enough to subject to real time imaging.
Okabe
Kohki
K
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 1130033 Japan.
Ikeda
Hisafumi
H
Harada
Yoshie
Y
Funatsu
Takashi
T
D016375:Antisense Elements (Genetics) / Q000737:chemistry*; D005057:Eukaryotic Cells / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D020135:Peptide Nucleic Acids / Q000737:chemistry; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*
D016375:Antisense Elements (Genetics); D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D012313:RNA