Shi Hua, Fan Xiaochun, Sevilimedu Aarti, Lis John T
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3742-6. doi: 10.1073/pnas.0607805104. Epub 2007 Feb 28.
Cellular regulatory networks are organized such that many proteins have few interactions, whereas a few proteins have many. These densely connected protein "hubs" are critical for the system-wide behavior of cells, and the capability of selectively perturbing a subset of interactions at these hubs is invaluable in deciphering and manipulating regulatory mechanisms. SELEX-generated RNA aptamers are proving to be highly effective reagents for inhibiting targeted proteins, but conventional methods generate one or several aptamer clones that usually bind to a single target site most preferred by a nucleic acid ligand. We advance a generalized scheme for isolating aptamers to multiple sites on a target molecule by reducing the ability of the preferred site to select its cognate aptamer. We demonstrate the use of this scheme by generating aptamers directed to discrete functional surfaces of the yeast TATA-binding protein (TBP). Previously we selected "class 1" RNA aptamers that interfere with the TBP's binding to TATA-DNA. By masking TBP with TATA-DNA or an unamplifiable class 1 aptamer, we isolated a new aptamer class, "class 2," that can bind a TBP.DNA complex and is in competition with binding another general transcription factor, TFIIA. Moreover, we show that both of these aptamers inhibit RNA polymerase II-dependent transcription, but analysis of template-bound factors shows they do so in mechanistically distinct and unexpected ways that can be attributed to binding either the DNA or TFIIA recognition sites. These results should spur innovative approaches to modulating other highly connected regulatory proteins.
细胞调控网络的组织方式是，许多蛋白质之间的相互作用较少，而少数蛋白质之间的相互作用较多。这些高度连接的蛋白质“枢纽”对于细胞的全系统行为至关重要，能够选择性地干扰这些枢纽处的一部分相互作用，对于解读和操纵调控机制具有极高价值。经SELEX技术产生的RNA适体已被证明是抑制靶向蛋白质的高效试剂，但传统方法产生的一个或几个适体克隆通常会结合到核酸配体最偏好的单个靶位点上。我们提出了一种通用方案，通过降低偏好位点选择其同源适体的能力，来分离针对靶分子上多个位点的适体。我们通过生成针对酵母TATA结合蛋白（TBP）离散功能表面的适体，展示了该方案的应用。此前我们筛选出了干扰TBP与TATA-DNA结合的“1类”RNA适体。通过用TATA-DNA或不可扩增的1类适体掩盖TBP，我们分离出了一种新的适体类别“2类”，它可以结合TBP-DNA复合物，并与另一种通用转录因子TFIIA的结合存在竞争。此外，我们表明这两种适体都能抑制RNA聚合酶II依赖的转录，但对模板结合因子的分析表明，它们是以机制上不同且出人意料的方式做到这一点的，这可归因于它们结合了DNA或TFIIA识别位点。这些结果应会促使人们采用创新方法来调控其他高度连接的调控蛋白。
