Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides.H erein, we report ahighly practical solution for reliable one-step selection of aptamers.W eintroduce partitioning by ideal-filter capillary electrophoresis (IFCE) in whichbinders and nonbinders move in the opposite directions.T he efficiency of IFCE-based partitioning reaches 10 9 ,w hichi st en million times higher than that of typical solid-phase partitioning methods.One step of IFCE-based partitioning is sufficient for the selection of ah igh-affinity aptamer pool for ap rotein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.Aptamers are oligonucleotides that can bind target molecules with high affinity and selectivity; [1] they find avariety of practical applications. [2] Aptamers are typically selected from random-sequence oligonucleotide libraries in ap rocess termed SELEX. [1][2][3] SELEX involves iterated rounds of incubation of the library with the target followed by partitioning of target-binding oligonucleotides (binders) from target-nonbinding oligonucleotides (nonbinders) and PCR amplification of all collected oligonucleotides until the binder-to-nonbinder ratio (B/N)r eaches ad esired value, preferably greater than unity.R emarkably,S ELEX fails to select binders in 70 %o fa ttempts. [4] This multi-round procedure is inherently prone to failure because PCR preferentially amplifies nonbinders,which are less structured oligonucleotides than binders and are,h ence,m ore easily accessible to polymerases. [5] As ar esult, SELEX enriches readily amplifiable nonbinders instead of binders if the efficiency of enriching binders in partitioning is lower than the efficiencyo fe nriching these nonbinders in PCR amplification. [6] An obvious solution to this daunting problem is increasing the efficiency of partitioning to the level at which its single step becomes sufficient for reaching the desired B/N. There have been several reports claiming one-step selection of aptamers. [7] However, neither of the suggested methods has been independently confirmed since their introduction in 2005-2012, thus,questioning at least their transferability and practicality and likely their reliability.H erein, we report on aq uantitatively validated, highly practical, and easily adoptable approach for one-step selection of aptamers.W eh ope that the new approach will be adopted and successfully used by many in the large and diverse in vitro selection community.This work was inspired by our understanding that there are two major reasons for the lack of ar obust and practical way of one-step selection of aptamers.T he first reason is methodological;while high efficiencies of partitioning are the implied goal, they are typically not measured and not used to guide developments or substantiate claims of one-s...

Partitioning of protein−DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here, we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE), which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of the protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinders must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for largesize protein targets while NECEEM should be the method of choice for small-size protein targets.

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.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is an affinity method for separating binder-target complexes from nonbinders by gel-free CE. NECEEM is a promising high-efficiency method for partitioning protein binders from nonbinders in DNA-encoded libraries (DEL), such binders are used as "hits" in drug development. It is important to be able to predict the efficiency of NECEEM-based partitioning, which is the efficiency of collecting binders while removing nonbinders for a specific protein and a specific DEL with a minimum of empirical information. Here, we derive and study the dependence of efficiency of NECEEM-based partitioning on electrophoretic mobilities of the protein and the DNA moiety in DEL compounds. Our derivation is based upon a previously found relation between the electrophoretic mobility of protein-binder complex and measured electrophoretic mobilities of the protein and unbound DEL and their estimated sizes. The derivation utilizes the assumption of Gaussian shapes of electrophoretic peaks and the approximation of the efficiency of partitioning by the background of nonbinders - a fraction of nonbinders, which elutes along with protein-binder complexes. Our results will serve as a guiding tool for planning the NECEEM-based partitioning of protein binders from non-binders in DELs. In particular, it can be used to estimate a minimum number of rounds of partitioning required for the desired level of DEL enrichment.

Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein‐bound oligonucleotides (binders) from protein‐unbound oligonucleotides (nonbinders). We have recently introduced ideal‐filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein‐binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 109, i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein‐binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid‐phase‐based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in‐depth IFCE's prospective in practical applications and fundamental studies.