Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.
We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.
Ota
N
N
National Institute for Advanced Interdisciplinary Research, and National Institute of Bioscience and Human Technology, Agency of Industrial Science & Technology, MITI, Tsukuba Science City 305, Japan.
Hirano
K
K
Warashina
M
M
Andrus
A
A
Mullah
B
B
Hatanaka
K
K
Taira
K
K
D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004735:Energy Transfer; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D015336:Molecular Probe Techniques; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry; D016337:RNA, Catalytic / Q000235:genetics; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000379:methods*
C106141:6-carboxytetramethylrhodamine; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D016337:RNA, Catalytic; D012235:Rhodamines; C024098:6-carboxyfluorescein; D004247:DNA