Nucleic Acids Research, Vol 26, Issue 3 735-743, Copyright © 1998 by Oxford University Press
N Ota, K Hirano, M Warashina, A Andrus, B Mullah, K Hatanaka and K Taira
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.
ARTICLES
Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids
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.
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