Characterization of anti-NF-kappaB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system.
Characterization of anti-NF-kappaB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system.
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DOI：
10.1093/nar/gkp670
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发表时间：
2009-10
影响因子：
14.9
通讯作者：
Maher LJ 3rd
中科院分区：
文献类型：
--
作者：
Wurster SE;Bida JP;Her YF;Maher LJ 3rd
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-κB p50和p65 RNA适体的体外选择和表征。我们现在描述这些适体在体外和体内的进一步表征。我们发现，在所有测试条件下，亚饱和浓度的某些抗p50 RNA适体促进与NF-κB p50四聚体的复合物形成，而抗p65 R1 RNA适体结合NF-κB二聚体。酵母三杂交RNA适体特异性研究证实了先前的体外结果，证实了抗p50和抗p65 R1 RNA适体分别对NF-κB p502和p652具有高度特异性。这些研究引入了一种新的T盒RNA转录物，其改善了来自四向RNA连接的RNA展示。抗p65 R1适体的突变揭示了耐受的取代，表明复杂的三级结构。我们描述了从酵母三杂交RNA文库中进行的体内选择，该文库含有在R1 SELEX过程早期存在的序列，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，让人想起抗p50。Y1在体外竞争性抑制NF-κB p652的DNA结合域。
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.
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DOI：
10.1073/pnas.0736013100
发表时间：
2003-04-01
影响因子：
11.1
作者：
Cassiday, LA;Maher, LJ
通讯作者：
Maher, LJ
影响因子：
4.8
作者：
Madrid, LV;Mayo, MW;Baldwin, AS
通讯作者：
Baldwin, AS
影响因子：
2.9
作者：
Cassiday, LA;Lebruska, LL;Maher, LJ
通讯作者：
Maher, LJ
影响因子：
5.3
作者：
CAPONIGRO, G;MUHLRAD, D;PARKER, R
通讯作者：
PARKER, R
影响因子：
2.9
作者：
King, DJ;Bassett, SE;Gorenstein, DG
通讯作者：
Gorenstein, DG