Fluorescence resonance energy transfer analysis of the degradation of an oligonucleotide protected by a very stable hairpin.
In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases. The FRET technique proved adequate for an in situ study of these protected antisense oligonucleotides in living cells.
Réfrégiers
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Laboratoire de Physicochimie Biomoléculaire et Cellulaire, URA CNRS 2056, Université, P. et M. Curie, Paris, France.
Laigle
A
A
Jollès
B
B
Chinsky
L
L
D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis / Q000379:methods
D005452:Fluoresceins; D009841:Oligonucleotides; D012235:Rhodamines; D003851:Deoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D019793:Fluorescein