Tian Y, Adya N, Wagner S, Giam C Z, Green M R, Ellington A D
Department of Chemistry, Indiana University, Bloomington 47405, USA.
RNA. 1995 May;1(3):317-26.
Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.
从随机序列库中分离出的核酸适配体通常已被证明可用于抑制核酸结合蛋白与其同源核酸之间的相互作用。为了开发也可用于研究蛋白质：蛋白质相互作用的试剂，我们利用体外筛选来寻找能够与人T细胞白血病病毒的反式激活蛋白Tax相互作用的RNA适配体。Tax通常不与核酸结合，而是通过与多种细胞转录因子相互作用来刺激转录，这些转录因子包括环磷酸腺苷反应元件结合蛋白（CREB）、核因子κB和血清反应因子（SRF）。从一个具有120个随机序列位置核心的超过10^13种不同RNA的库开始，选择能够与Tax共同保留在硝酸纤维素滤膜上的RNA。经过五个循环的筛选和扩增后，只剩下一种核酸。发现这种适配体以高亲和力和特异性结合Tax，并且可以破坏Tax与核因子κB之间的复合物形成，但不能破坏Tax与SRF之间的复合物形成。我们的适配体探针对蛋白质：蛋白质相互作用的不同影响提示了一种关于Tax蛋白表面转录因子结合位点如何组织的模型。该模型与来自其他各种研究的数据一致。
