Ideal‐filter capillary electrophoresis: A highly efficient partitioning method for selection of protein binders from oligonucleotide libraries

Abstract

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.

Challenging Bioanalyses with Capillary Electrophoresis

“…The process of selection and enrichment is simplified when the separation between nonbinding and binding sequences is increased. A new technique called ideal filter capillary electrophoresis (IFCE) was introduced during the time frame of this Review. , IFCE overcomes limitations of CE-SELEX and NECEEM because the target–binder complex and nonbinding sequence migrate in opposite directions, which reduces the amount of nonbinding sequences present in the fraction containing the binding sequences. This is achieved by adjusting the ionic strength of the running buffer to decrease the electroosmotic flow to be within a specific range.…”

Capillary electrophoresis‐based methodology for screening of oligonucleotide aptamers

“…The PCR amplification of the ssDNA-enriched pool obtained after every round of screening is essential in selecting aptamers by NECEEM. It is very difficult to obtain aptamers through single-round screening because their abundance in the random library is only 10 −6 -10 −9 (Le, Krylova, Kanoatov, et al, 2019;Le, Krylova, & Krylov, 2019b). Many unbound ssDNAs can leak out to the bound ssDNAs during partitioning, forming the background of free ssDNAs.…”

“…Lisi et al obtained its aptamer after three rounds of non-SELEX screening combined with high-throughput sequencing (Lisi et al, 2018). The aptamer can recognize not only whole τ-441 but also τ-381, τ-352 and τ-383 (Le, Krylova, Kanoatov, et al, 2019;Le, Krylova, & Krylov, 2019b strength of running buffer. They found that complex velocity > 0 > free ssDNA velocity and k B /k N > 1.7 × 10 9 could be realized and the condition of one-step screening could be satisfied through adding 100 mM NaCl into running buffer when using MutS (pI = 6) as target.…”

Affinity Capillary Electrophoresis: A Critical Review of the Literature from 2018 to 2020

“…The measured K d,app was 40 nM, which could be achieved but with three rounds of CE-SELEX. Several follow up papers on IFCE have already been published describing further details and equilibrium constant and rate constant determinations . Overall, IFCE is an exciting technique for high efficiency selection of aptamers.…”