An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.
An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.
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DOI：
10.1021/ja807609m
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发表时间：
2009-02-18
影响因子：
15
通讯作者：
Yu J
中科院分区：
文献类型：
--
作者：
Lee SJ;Hyun S;Kieft JS;Yu J
发夹RNA基序是最常见的二级结构之一，并且经常被治疗剂靶向。设计了一种具有7个赖氨酸和8个亮氨酸残基的两亲性肽及其衍生物作为RNA发夹结构的配体。我们假设这些两亲肽的疏水性富含亮氨酸和亲水性富含赖氨酸的球体的变化将与发夹RNA靶产生额外的吸引力相互作用。一系列丙氨酸扫描的肽进行了探测，以确定最有影响力的赖氨酸残基的亲水球。测定了这些修饰肽与几种发夹如HIV的RRE、TAR、HCV的IRES的短发夹和rRNA的16S A位点茎的发夹的结合亲和力。由于来自HCV的IRES的发夹对丙氨酸扫描的肽的初始系列最敏感，因此研究肽中的进一步变化如何影响结合的研究采用了IRES发夹。接下来，重要的赖氨酸残基被较短链的胺（如鸟氨酸）取代，以将肽更深地置于发夹沟中。在少数情况下，观察到含有特异性定位的较短胺侧链的肽的结合提高了70倍。为了进一步探索疏水球改变所带来的结合亲和力的变化，引入色氨酸残基代替亮氨酸。在特定位置具有色氨酸的一些肽也显示出70倍的结合亲和力改善。最后，双突变体肽纳入两个具体定位的较短的胺侧链在亲水性区域和色氨酸残基在疏水性区域的合成。观察到含有简单双重修饰的肽的结合亲和力低80倍，并且它们的结合特异性增加40倍。这项工作的结果提供了有关可用于制备强烈和选择性靶向发夹RNA的肽的策略的重要信息。具体而言，研究结果表明，如果RNA靶标具有深凹槽，则可以获得针对某些发夹RNA的定制的两亲性肽配体，其中肽的疏水性和亲水性球体相互作用。
The hairpin RNA motif is one of the most frequently observed secondary structures and is often targeted by therapeutic agents. An amphiphilic peptide with seven lysine and eight leucine residues and its derivatives were designed for use as ligands against RNA hairpin motifs. We hypothesized that variations in both the hydrophobic leucine-rich and hydrophilic lysine-rich spheres of these amphiphilic peptides would create extra attractive interactions with hairpin RNA targets. A series of alanine-scanned peptides were probed to identify the most influential lysine residues in the hydrophilic sphere. The binding affinities of these modified peptides with several hairpins, such as RRE, TAR from HIV, a short hairpin from IRES of HCV, and a hairpin from the 16S A-site stem from rRNA, were determined. Since the hairpin from IRES of HCV was the most susceptible to the initial series of alanine-scanned peptides, studies investigating how further variations in the peptides effect binding employed the IRES hairpin. Next, the important Lys residues were substituted by shorter chain amines, such as ornithine, to place the peptide deeper into the hairpin groove. In a few cases, a 70-fold improved binding was observed for peptides that contained the specifically located shorter amine side chains. To further explore changes in binding affinities brought about by alterations in the hydrophobic sphere, tryptophan residues were introduced in place of leucine. A few peptides with tryptophan in specific positions also displayed 70-fold improved binding affinities. Finally, double mutant peptides incorporating both specifically located shorter amine side chains in the hydrophilic region and tryptophan residues in the hydrophobic region were synthesized. The binding affinities of peptides containing the simple double modification were observed to be 80 times lower, and their binding specificities were increased 40-fold. The results of this effort provide important information about strategies that can be used to prepare peptides that both strongly and selectively target hairpin RNAs. Specifically, the findings indicate that tailor-made amphiphilic peptide ligands against certain hairpin RNAs can be obtained if the RNA target possesses a deep groove in which both the hydrophobic and hydrophilic spheres of the peptide interact.
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