Background

Aptamers are single strands of synthetic DNA or RNA described in 1990 (Ellington and Szostak, 1990; Tuerk and Gold, 1990) with a distinct 3D geometry, which is a manifestation of their sequences. Different sequences allow the synthesis of aptamers, which can bind to an array of molecules ranging from small molecules to large proteins. This allowed aptamers to emerge as the rivals to conventional antibodies, which are limited to proteins as their targets due to structural limitations and can not be raised against high-risk pathogens as these usually kill the host much earlier than the time required to generate high affinity antibodies. Aptamers are generated in in-vitro setups and thus can effectively be used for high-risk pathogens. SELEX (Systematic Evolution of Ligands by EXponential enrichment) is an iterative process involving repeated exposures and separation leading to screening of a pool with dissociation constants in low nM ranges. Several variations of SELEX have been proposed which use a variety of stationary matrices including capillary electrophoresis SELEX, affinity chromatography based SELEX, magnetic bead SELEX, in-vivo SELEX, FACS SELEX and Microfluidics based SELEX, etc. These methods present various advantages over others but the number of SELEX rounds are consistently high.

A standard SELEX method has four major steps; exposure of random sequence single stranded nucleotide oligo library to target, binding of oligos to target molecule, selection of binders and removal of non-binding oligos, amplification of the binder fraction and portioning of the amplicon to single strand. These steps are iteratively performed till pool of high binding aptamer are screened out from the library. The oligonucleotide library consists of a random base-sequence flanked on both ends by primer binding sites, which aids in amplification and enrichment (Safeh et al., 2010; Kim et al., 2013).

We report a microtiter plate-based Cell-SELEX method, which can be employed over a wide number of molecules and cell types (Figure 1). The advantage proposed by the method is uniquely low number of rounds, which are the outcome of large surface area, higher retention times and a novel superior bio-probe based partitioning process (Priyanka et al., 2014).


Figure 1. Concise Protocol Schematic. A step-by-step flowchart showing the various steps of Cell-SELEX. (Modified from author’s work Kaur et al., 2017)