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Antisense activity detection by inhibition of fluorescence resonance energy transfer
Author(s) -
BenítezHess M. L.,
DiPaolo J. A.,
AlvarezSalas L. M.
Publication year - 2004
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/bio.763
Subject(s) - förster resonance energy transfer , green fluorescent protein , microbiology and biotechnology , mutant , in vivo , molecular beacon , chemistry , nucleic acid , biophysics , fluorescence , biology , biochemistry , gene , oligonucleotide , genetics , physics , quantum mechanics
Abstract Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV‐16)‐associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS‐ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of uorescence resonance energy transfer (FRET) to measure the interaction of AS‐ODNs with HPV‐16 target nt 410–445, using variants of the green uorescent protein (GFP). An optimal FRET‐producing pair was selected with GFP as the donor and yellow uorescent protein (YFP) as the acceptor molecule. Hybridization of AS‐ODNs with a chimaeric mRNA containing the antisense target site anked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no signicant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target‐specic and mutant AS‐ODNs, suggested a specic effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins conrming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS‐ODNs activity in vivo . Copyright © 2004 John Wiley & Sons, Ltd.