Ideal‐Filter Capillary Electrophoresis (IFCE) Facilitates the One‐Step Selection of Aptamers

Abstract

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...

Spherical-Shape Assumption for Protein–Aptamer Complexes Facilitates Prediction of Their Electrophoretic Mobility

“…8 It will also facilitate rational selection of the number of fractions to be blindly collected in the IFCE-based partitioning. 9 Overall, this mobility predictor will be an indispensable tool in rational design of CE-based aptamer selection. Table S1.…”

“…The aptamer pool for experiments with streptavidin was generated from ssDNA library that contained a random region (N) of 40 nucleobases, using IFCE. 9 The sequence of the library was as follows: 5′-/Alex488N/CTA CGG TAA ATC GGC AGT CAN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN ATC TGA AGC ATA GTC CAG GC-3′. Tris-acetate (50 mM final concentration, pH 8.2) was used as both a running buffer and a dilution buffer for all CE experiments with MutS, AlkB, and streptavidin.…”

“…8 In recently introduced ideal-filter CE (IFCE), the complexes move towards the capillary outlet while unbound DNA moves backwards to the inlet. 9 The efficiency of IFCEbased partitioning reaches 10 9 , which is presumed to be sufficient for reliable selection of aptamers in a single step of partitioning.…”

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

“…Finally, we demonstrated one‐step selection of a high‐affinity aptamer pool to a protein target (MutS protein) to confirm that the estimated efficiency of 10 9 required for one‐step selection was correct. Our first report on IFCE has been recently published . Here, we elaborate on the justification for IFCE development and important electrophoretic aspects of the method.…”

Selection Technologies and Applications of Nucleic Acid Aptamers

“…Krylov and coworkers developed non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-SELEX, 6 equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM)-SELEX, 7 and ideal-filter CE (IFCE)-SELEX. 8 Saito and coworkers reported polymer-enhanced capillary transient isotachophoresis (PectI)-SELEX for cell-binding aptamers. 9,10 Wakui et al discovered a thrombin-binding aptamer, which had 10 to 20-fold higher anticoagulation activity than other earlier thrombin-binding aptamers in vitro studies, by microbeads-assisted CE (MACE)-SELEX.…”