Expanding the chemical diversity of aptamers remains an important thrust in the field in order to increase their functional potential. Previously, our group developed LOOPER, which enables the incorporation of up to 16 unique modifications throughout a ssDNA sequence, and applied it to the in vitro evolution of thrombin binders. As LOOPER-derived highly modified nucleic acids polymers are governed by two interrelated evolutionary variables, namely, functional modifications and sequence, the evolution of this polymer contrasts with that of canonical DNA. Herein we provide in-depth analysis of the evolution, including structure-activity relationships, mapping of evolutionary pressures on the library, and analysis of plausible evolutionary pathways that resulted in the first LOOPER-derived aptamer, TBL1. A detailed picture of how TBL1 interacts with thrombin and how it may mimic known peptide binders of thrombin is also proposed. Structural modeling and folding studies afford insights into how the aptamer displays critical modifications and also how modifications enhance the structural stability of the aptamer. A discussion of benefits and potential limitations of LOOPER during in vitro evolution is provided, which will serve to guide future evolutions of this highly modified class of aptamers.

中文翻译：

---

从体外进化中分离出功能多样的适体的进化结果。

扩大适体的化学多样性仍然是该领域中的重要目的，以增加其功能潜力。以前，我们的小组开发了LOOPER，它能够在一个ssDNA序列中整合多达16个独特的修饰，并将其应用于凝血酶结合物的体外进化。由于LOOPER衍生的高度修饰的核酸聚合物受两个相互关联的进化变量（即功能修饰和序列）支配，因此该聚合物的进化与规范DNA的进化相反。本文中，我们提供了进化的深入分析，包括结构-活性关系，库上进化压力的作图以及可能产生第一个LOOPER适体TBL1的合理进化途径的分析。还提出了TBL1如何与凝血酶相互作用以及如何模仿凝血酶的已知肽结合剂的详细图片。结构建模和折叠研究为了解适体如何显示关键修饰以及修饰如何增强适体的结构稳定性提供了见识。提供了关于LOOPER在体外进化过程中的益处和潜在局限性的讨论，这将有助于指导这种高度修饰的适体类别的未来进化。