Benítez-Hess M L, DiPaolo J A, Alvarez-Salas L M
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.
Luminescence. 2004 Mar-Apr;19(2):85-93. doi: 10.1002/bio.763.
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.
使用反义核酸来调节特定基因的表达是治疗人乳头瘤病毒16型（HPV - 16）相关宫颈癌的一种有前景的方法。可以理解的是，评估合成反义寡脱氧核苷酸（AS - ODNs）或核酶的体内性能对于开发有效的反义工具至关重要。在此我们报告了一种基于荧光共振能量转移（FRET）抑制的细菌报告系统的使用，该系统利用绿色荧光蛋白（GFP）的变体来测量AS - ODNs与HPV - 16靶标核苷酸410 - 445的相互作用。选择了以GFP作为供体和黄色荧光蛋白（YFP）作为受体分子的最佳产生FRET的对。AS - ODNs与含有由GFP变体侧翼的反义靶位点的嵌合mRNA杂交导致FRET效应的抑制。使用不同的接头表明接头的氨基酸含量对FRET效应没有显著影响。通过用硫代磷酸化的靶标特异性和突变AS - ODNs进行RNaseH测定来测试反义可及性，表明对嵌合mRNA有特异性作用。FRET抑制测量与截短蛋白的存在相关，证实了对靶标的真正反义活性。因此，FRET抑制可用于直接测量体内AS - ODNs的活性。
