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Characterization of anti-NF-κB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system

机译:体外和酵母三杂交系统中抗NF-κBRNA适体结合特异性的表征

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摘要

RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-κB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-κB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-κB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-κB p502 and p652, respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-κB p652 in vitro.
机译:RNA适体为竞争性抑制DNA结合转录因子提供了一种潜在的治疗方法。在以前的报告中,我们描述了抗NF-κBp50和p65 RNA适体的体外选择和表征。现在我们描述体外和体内这些适体的进一步表征。我们显示某些抗p50 RNA适体的亚饱和浓度促进与NF-κBp50四聚体形成复合物,而抗p65 R1 RNA适体在所有测试条件下均与NF-κB二聚体结合。酵母三杂交RNA适体特异性研究证实了先前的体外结果,证实抗p50和抗p65 R1 RNA适体分别对NF-κBp502和p652具有高度特异性。这些研究介绍了一种新型的T型盒式RNA转录本,可改善四向RNA连接处的RNA展示。抗p65 R1适体的诱变揭示了可耐受的取代,表明其复杂的三级结构。我们描述了从酵母三杂种RNA文库的体内选择,该文库包含在R1 SELEX过程早期发现的序列,以鉴定新型抗p65 RNA适体,称为Y1和Y3。这些适体似乎是紧凑的凸起发夹,让人联想到抗p50。 Y1在体外竞争性抑制NF-κBp652的DNA结合结构域。

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