title: Antisense activity detection by inhibition of fluorescence resonance energy transfer.
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 fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked 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 significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.
authors.affiliation: Laboratorio de Terapia Genica, Departamento de Genetica y Biologia Molecular, Centro de Investigacion y de Estudios Avanzados, Av. IPN 2508, Mexico D.F. 07360, Mexico.
authors.forename: M L
authors.initials: ML
authors.lastname: Benítez-Hess
authors.forename: J A
authors.initials: JA
authors.lastname: DiPaolo
authors.forename: L M
authors.initials: LM
authors.lastname: Alvarez-Salas
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D015344:DNA Probes, HPV / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D015344:DNA Probes, HPV; D008164:Luminescent Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins