Designed arginine-rich RNA-binding peptides with picomolar affinity
Designed arginine-rich RNA-binding peptides with picomolar affinity
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DOI：
10.1021/ja026610b
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发表时间：
2002-09-18
影响因子：
15
通讯作者：
Roberts, RW
中科院分区：
文献类型：
--
作者：
Austin, RJ;Xia, TB;Roberts, RW
富含精氨酸的肽基序（ARMs）能够结合独特的RNA结构，在转录、翻译、RNA运输和RNA包装中起着至关重要的作用。转录抗终止所需的噬菌体ARMs与具有诱导α-螺旋结构的不同boxB RNA发夹序列结合。对取自羔羊样噬菌体的ARM的表征表明，在生理浓度的一价阳离子下，P22噬菌体模型-抗终止复合物（P22N21 - P22boxB）在游离溶液中的解离常数为200±56 pM，明显强于之前通过凝胶迁移位移和聚丙烯酰胺凝胶共电泳测定的解离常数，比已知最紧密的天然ARM - RNA在生理盐下的相互作用强2个数量级。在这里，我们使用互反设计方法来增强两个单独的α-螺旋ARM−RNA相互作用的结合亲和力；一个来自天然λ噬菌体抗终止复合体，另一个通过mRNA展示选择实验分离到靶向boxB RNA。
Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB RNA hairpin sequences with a characteristic induced α-helical structure. Characterization of ARMs from lambdoid phages reveals that the dissociation constant of the P22 bacteriophage model−antitermination complex (P22N21−P22boxB) is 200 ± 56 pM in free solution at physiologic concentrations of monovalent cation, significantly stronger than previously determined by gel mobility shift and polyacrylamide gel coelectophoresis, and 2 orders of magnitude stronger than the tightest known native ARM−RNA interaction at physiological salt. Here, we use a reciprocal design approach to enhance the binding affinity of two separate α-helical ARM−RNA interactions; one derived from the native λ phage antitermination complex and a second isolated using mRNA display selection experiments targeting boxB RNA.