Le An T H, Krylova Svetlana M, Beloborodov Stanislav S, Wang Tong Y, Hili Ryan, Johnson Philip E, Li Feng, Veedu Rakesh N, Belyanskaya Svetlana, Krylov Sergey N
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada.
Department of Chemistry and Centre for Biotechnology, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, Ontario L2S 3A1, Canada.
Anal Chem. 2021 Apr 6;93(13):5343-5354. doi: 10.1021/acs.analchem.1c00601. Epub 2021 Mar 25.
Screening molecular libraries for ligands capable of binding proteins is widely used for hit identification in the early drug discovery process. Oligonucleotide libraries provide a very high diversity of compounds, while the combination of the polymerase chain reaction and DNA sequencing allow the identification of ligands in low copy numbers selected from such libraries. Ligand selection from oligonucleotide libraries requires mixing the library with the target followed by the physical separation of the ligand-target complexes from the unbound library. Cumulatively, the low abundance of ligands in the library and the low efficiency of available separation methods necessitate multiple consecutive rounds of partitioning. Multiple rounds of inefficient partitioning make the selection process ineffective and prone to failures. There are continuing efforts to develop a separation method capable of reliably generating a pure pool of ligands in a single round of partitioning; however, none of the proposed methods for single-round selection have been universally adopted. Our analysis revealed that the developers' efforts are disconnected from each other and hindered by the lack of quantitative criteria of selection quality assessment. Here, we present a formalism that describes single-round selection mathematically and provides parameters for quantitative characterization of selection quality. We use this formalism to define a universal strategy for development and validation of single-round selection methods. Finally, we analyze the existing partitioning methods, the published single-round selection reports, and some pertinent practical considerations through the prism of this formalism. This formalism is not an experimental protocol but a framework for correct development of experimental protocols. While single-round selection is not a goal by itself and may not always suffice selection of good-quality ligands, our work will help developers of highly efficient selection approaches to consolidate their efforts under an umbrella of universal quantitative criteria of method development and assessment.
筛选能够结合蛋白质的配体的分子文库在早期药物发现过程中被广泛用于寻找活性分子。寡核苷酸文库提供了非常高的化合物多样性，而聚合酶链反应和DNA测序的结合则允许从这样的文库中鉴定低拷贝数的配体。从寡核苷酸文库中选择配体需要将文库与靶标混合，然后将配体-靶标复合物与未结合的文库进行物理分离。总的来说，文库中配体的低丰度和现有分离方法的低效率使得需要进行多轮连续的分配。多轮低效的分配使得选择过程无效且容易失败。人们一直在努力开发一种能够在一轮分配中可靠地产生纯配体库的分离方法；然而，所提出的单轮选择方法都没有被普遍采用。我们的分析表明，开发者的努力相互脱节，并且由于缺乏选择质量评估的定量标准而受到阻碍。在这里，我们提出一种形式主义，它用数学方法描述单轮选择，并提供用于定量表征选择质量的参数。我们使用这种形式主义来定义单轮选择方法开发和验证的通用策略。最后，我们通过这种形式主义的视角分析现有的分配方法、已发表的单轮选择报告以及一些相关的实际考虑因素。这种形式主义不是一个实验方案，而是正确开发实验方案的框架。虽然单轮选择本身不是一个目标，并且可能并不总是足以选择高质量的配体，但我们的工作将有助于高效选择方法的开发者在方法开发和评估的通用定量标准的框架下整合他们的努力。
