How to Develop and Prove High-Efficiency Selection of Ligands from Oligonucleotide Libraries: A Universal Framework for Aptamers and DNA-Encoded Small-Molecule Ligands

Abstract

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.

DNA aptamer selection for SARS-CoV-2 spike glycoprotein detection

“…To increase SELEX stringency, the resulting partitioned pool (1R) was used for two subsequent IFCE partition rounds (2R and 3R) with decreasing S protein concentrations (50 and 25 nM, respectively). This method was modified by performing extra selection rounds to increase strong binders, reduce the amount of non-binders and facilitate NGS enrichment analysis [ 54 ]. Binding assays showed that the M2 pool and 2R had differential binding affinities for S protein.…”

Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis

“…This knowledge can also help validate the results of single-round aptamer selection. 18 We found no reports on a quantitative study dedicated to what k N and k B depend on and how. The lack of such studies is arguably the major reason for aptamer selection still being more an art than science.…”

“…Partitioning is evidently a key step in aptamer selection increasing the efficiency of partitioning allows completion of aptamer selection in fewer rounds and can help avoid selection failures. 18 Partitioning can be conceptually presented as a physical filter that lets binders through but stops nonbinders (Figure 1a). It can then be described quantitatively using "transmittance".…”

Evolution of the Selection Methods of DNA-Encoded Chemical Libraries

“…30 However, these methods require protein tagging/modification and special library design and resynthesis. The Krylov group pioneered an approach to directly separate the target-ligand complex from the nonbinders with kinetic capillary electrophoresis (KCE; Figure 1c), 31 but it has only been tested with model systems.…”