Selection of DNA aptamers with two modified bases.
Selection of DNA aptamers with two modified bases.
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DOI：
10.1073/pnas.1615475114
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发表时间：
2017-03-14
影响因子：
11.1
通讯作者：
Janjic N
中科院分区：
文献类型：
--
作者：
Gawande BN;Rohloff JC;Carter JD;von Carlowitz I;Zhang C;Schneider DJ;Janjic N
适体现在普遍用作广泛应用的结合剂。天然（未修饰的）DNA和RNA适体具有比基于蛋白质的配体（例如抗体）少得多的化学多样性，限制了它们的实用性。具有单一化学修饰的适体有助于弥合这种多样性差距。我们报告了选择和鉴定具有两种多样性增强化学修饰的适体，其结合并抑制前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9），这是一种代表性的人类治疗性蛋白质靶标。添加第二修饰，特别是在某些成对组合中，导致亲和力、配体效率、表位覆盖率、代谢稳定性和抑制活性的显著改善。广泛化学功能化的适体具有成为下一代基于核酸的配体的潜力。包含DNA和RNA适体的核碱基提供比基于蛋白质的配体少得多的化学多样性，限制了它们的通用性。最近，仅在修饰的适体的四个碱基之一上引入新的官能团，大大提高了识别蛋白质靶点的核酸配体的成功率。在这里，我们探索的好处，通过选择修饰的DNA适体，包含两个嘧啶碱基上的氨基酸样修饰的物理化学多样性的额外增强。使用前蛋白转化酶枯草杆菌蛋白酶/kexin 9型作为代表性的蛋白质靶标，我们确定了特定的成对组合的修饰，导致更高的亲和力，代谢稳定性，和抑制效力相比，适配体与单一的修改。这种双重修饰的适体也更可能以较短的序列编码，并且比具有单一修饰的适体更频繁地占据非重叠表位。这些高度修饰的DNA适体在研究、诊断和治疗应用中具有广泛的用途。
Aptamers are now used ubiquitously as binding agents for a broad range of applications. Natural (unmodified) DNA and RNA aptamers have considerably less chemical diversity than protein-based ligands such as antibodies, limiting their utility. Aptamers possessing a single chemical modification have helped bridge this diversity gap. We report the selection and identification of aptamers with two diversity-enhancing chemical modifications that bind and inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9), a representative human therapeutic protein target. The addition of a second modification, especially in certain pairwise combinations, resulted in significant improvements in affinity, ligand efficiency, epitope coverage, metabolic stability, and inhibitory activity. Extensively chemically functionalized aptamers have the potential to become the next generation of nucleic-acid–based ligands. The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.