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. Herein, we report a highly practical solution for reliable one‐step selection of aptamers. We introduce partitioning by ideal‐filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE‐based partitioning reaches 109, which is ten million times higher than that of typical solid‐phase partitioning methods. One step of IFCE‐based partitioning is sufficient for the selection of a high‐affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

Comprehensive analysis of drugs to treat SARS‑CoV‑2 infection: Mechanistic insights into current COVID‑19 therapies (Review)

“…At first instance, aptamer-mediated inhibition of ACE2 binding and TMPRSS2 activity, either solely or as a combination therapy, presents a rational means of intervention to prevent viral adhesion and cellular entry, similar to that proposed by small molecule inhibitors. Although cost of goods, chemistry, manufacturing and control requirements are significantly higher for this chemotherapeutic class vs small molecule drugs, rapid hit generation by modern aptamer development methods (210,211) and established orthogonal aptamer optimization strategies can deliver clinical candidates compatible with scalable manufacturing pertinent to pandemic outbreaks, in timelines comparable or better than antibody biotherapeutics. Thus, in the event of complete failure across the vast number of repurposed medication trials described in this review, a second wave of aptamer-based treatments could be ready for subsequent clinical testing.…”

SELEX-based DNA Aptamer Selection: A Perspective from the Advancement of Separation Techniques

“…To minimize the number of selection rounds, single-round selection has been proposed: IFCE, 23 FFE, 67 and CMACS 68 for proteins; PectI 30,31 for bacterial and cancer cells; and MonoLEX 95 for viruses. A common property of these methods is their very high PE.…”

“…[111][112][113] However, the compatibility of separation techniques used to acquire these functional aptamers is unclear, and it could be expected that unique separation modes could be identified to extract unique aptamers in the future. CE-SELEX, [15][16][17]61 non-SELEX, [18][19][20][21] Lp-CE-SELEX, 22 IFCE selection, 23 ss-CE-SELEX, 24,25 FCE-SELEX, 26 ctITP-SELEX, 28,29,62 80 GO-SELEX, [81][82][83][84]86 MRGO-SELEX, 85 SWCNTs-SELEX 87 ➢ FACS-based selection FACS-SELEX, 88,89 PD-SELEX, 92,93 Click-PD-SELEX 94 ➢ Other appraoches SOMAMer, 39,40 MonoLEX, 95 Sol-gel SELEX, 96 AFM-SELEX 97,98 ➢ Cloning ➢ High throughput sequencing (HTS) 29,37,54,[59][60][61][62] 1. Randomized ssDNA library ➢ Modified DNA library SOMAmer, [38][39][40] ...…”

“…Recently, Le et al reported a separation mode using CE in which each of complexed and nonbinding ssDNA (and ssDNA dissociated from the complex) migrates in opposite directions because of appropriate pressure applied to the capillary, termed ideal-filter capillary electrophoresis (IFCE). 23 With this method, a high-affinity DNA pool was successfully obtained by one step (single round) selection for the MutS protein. The efficiency of partitioning was shown to be 10 9 partition efficiency (PE; the ratio of the number of DNA served for selection to the number of partitioned DNA) and is the highest value reported so far; a PE of 10 2 is typical for solid-phase partitioning methods.…”

Study of molecular interactions by nonequilibrium capillary electrophoresis of equilibrium mixtures: Originations, developments, and applications

“…NECEEM represents a straightforward and robust technique for studying the interactions between proteins and DNA; therefore, partitioning protein-DNA complexes (binders) from protein-unbound DNA (nonbinders) is a particularly important step. Le et al presented NECEEM and ideal-filter capillary electrophoresis (IFCE) [55] as CEbased partitioning modes to evaluate the dependence of the transmittance of binders and nonbinders on the molecular weight of the protein target [56]. Here, the partitioning step is conceptually characterized by the transmittance of the binders and nonbinders.…”