Molecular tryst peeping: detection of interactions between nonlabeled nucleic acids by fluorescence resonance energy transfer.
We have developed a new method for monitoring the interactions between nonlabeled RNAs that involves detection of fluorescence resonance energy transfer (FRET) between two DNA probes with different fluorescent label. The sequences of the probes are complementary to those of the RNAs. In this study, we examined the interaction between a portion of the LTR RNA of HIV-1 and the corresponding antisense RNA. The antisense RNA was designed not to bind to the fluorescent DNA without prior hybridization to the target RNA. A mixture of RNAs and DNA probes with fluorescent labels was fractionated by electrophoresis on a nondenaturing polyacrylamide gel and then the gel was analyzed with a fluorescence imaging analyzer. FRET was observed only in the presence of target RNA, antisense RNA, and both of the fluorescent DNA probes. This strategy should be useful for the detection of interactions between nucleic acids that cannot be subjected to chemical modification, such as RNA transcripts inside cells.
Ota
N
N
Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku, Yokohama, 223-8522, Japan.
Sato
T
T
Taira
K
K
Ohkawa
J
J
D001483:Base Sequence; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry* / Q000378:metabolism*; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D015342:DNA Probes; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D016372:RNA, Antisense; D012367:RNA, Viral