SELEX Modifications and Bioanalytical Techniques for Aptamer–Target Binding Characterization

Abstract

The quest to improve the detection of biomolecules and cells in health and life sciences has led to the discovery and characterization of various affinity bioprobes. Libraries of synthetic oligonucleotides (ssDNA/ssRNA) with randomized sequences are employed during Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select highly specific affinity probes called aptamers. With much focus on the generation of aptamers for a variety of target molecules, conventional SELEX protocols have been modified to develop new and improved SELEX protocols yielding highly specific and stable aptamers. Various techniques have been used to analyze the binding interactions between aptamers and their cognate molecules with associated merits and limitations. This article comprehensively reviews research advancements in the generation of aptamers, analyses physicochemical conditions affecting their binding characteristics to cellular and biomolecular targets, and discusses various field applications of aptameric binding. Biophysical techniques employed in the characterization of the molecular and binding features of aptamers to their cognate targets are also discussed.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

“…However, it is interesting to note that despite the large number of aptamers reported to date, only a small subset of them has been characterized in detail. A number of biophysical techniques have been applied to determine binding kinetics and affinity values including stopped-flow fluorescence spectroscopy (Wickiser et al, 2005 ; Lang et al, 2007 ), equilibrium filtration (Huizenga and Szostak, 1995 ), gel-shift assays (Smith et al, 2009 ) and label-free methods such as isothermal titration calorimetry (ITC) (Lin et al, 2008 ; Sokoloski et al, 2012 ), kinetic ITC (Burnouf et al, 2012 ), surface-plasmon resonance (SPR) (Polonschii et al, 2010 ) and microfluidic SELEX (Tan et al, 2016 ). Selecting one of these methods depends on the specific aptamer under study and the required sensitivity as recently reviewed by DeRosa and coworkers (McKeague et al, 2015 ).…”

Peptides, Antibodies, Peptide Antibodies and More

“…A much-used method is SELEX (systematic evolution of ligands by exponential enrichment), which amplifies aptamers from random libraries by repetitive rounds of affinity selection (panning) combined with PCR amplification. However, aptamers amplified by this method usually requires further optimization in order to become practically useful, which is also testified by the relatively low number of currently useful aptamers [170,178]. One aptamer, which has found clinical use, is Pegaptanib, which is a vascular endothelial growth factor (VEGF)-targeting aptamer (Figure 3) used for treatment of various forms of pathological ocular neovascularization [166].…”

Development of Cell-Specific Aptamers: Recent Advances and Insight into the Selection Procedures

“…Flow cytometry is a laser-based method capable of characterizing physical and chemical properties of molecules with high reproducibility and accuracy [ 33 ]. Therefore, it has become one of the most common methods in characterizing aptamer binding affinities by passing a stream of single cell suspension through a detection apparatus in single file to detect tagged cells within a population.…”