Denton Kyle E, Krusemark Casey J
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University and Purdue University Center for Cancer Research, 575 Stadium Mall Dr., West Lafayette, IN, USA 47906.
Medchemcomm. 2016 Oct 1;7(10):2020-2027. doi: 10.1039/C6MD00288A. Epub 2016 Aug 2.
Achieving sufficient enrichment of ligands from DNA-encoded libraries for detection can be difficult, particularly for low affinity ligands within highly complex libraries. To address this challenge, we present an approach for crosslinking DNA-linked ligands to target proteins using electrophilic or photoreactive groups. The approach involves the teathering of a ssDNA oligonucleotide to a DNA-encoded molecule to enable attachment of a reactive group post-synthetically via DNA hybridization. Crosslinking traps ligand-protein complexes while in solution and allows for stringent washing conditions to be applied in the subsequent purification. Five reactive groups (tosyl, NHS ester, sulfonyl fluoride, phenyl azide, and diazirine) were tested for crosslinking efficiency and specificity with three DNA-linked ligands to their target proteins. In a model selection, crosslinking resulted in improved enrichment of both high and a low affinity ligands in comparison to a selection with a solid-phase immobilized protein.
从DNA编码文库中实现足够的配体富集以进行检测可能很困难，特别是对于高度复杂文库中的低亲和力配体。为应对这一挑战，我们提出了一种使用亲电或光反应性基团将DNA连接的配体与靶蛋白交联的方法。该方法涉及将单链DNA寡核苷酸连接到DNA编码分子上，以便通过DNA杂交在合成后连接反应性基团。交联在溶液中捕获配体-蛋白质复合物，并允许在后续纯化中应用严格的洗涤条件。测试了五个反应性基团（甲苯磺酰基、N-羟基琥珀酰亚胺酯、磺酰氟、苯基叠氮化物和重氮甲烷）与三种DNA连接的配体与其靶蛋白的交联效率和特异性。在模型筛选中，与使用固相固定化蛋白的筛选相比，交联导致高亲和力和低亲和力配体的富集均得到改善。
