Abstract
Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays.
Highlights
Aptamers recognize and bind to their targets through 3-dimensional shapes and various physiochemical interactions, similar to antibody binding mechanisms
Bruno reasoned that substitution of a DAP for the adenine proximal to I and/or T would either lead to an additional hydrogen bond or result in an additional repulsive force between the extra amine group’s electron pair in DAP and the highly electronegative polarized oxygen in the hydroxyl group of threonine, providing a means for discrimination and greater selectivity. While this approach only produced about a 20% greater binding of the modified aptamer to the prostate specific antigen (PSA) I-variant by enzyme-linked aptamer sorbent assay (ELASA) (Figure 7), this suggested that the aptamer-peptide binding was perturbed in that region by an increased repulsive force between DAP and Pragmatic Methods for Optimizing Aptamer Performance threonine of the PSA T-variant
Using the aforementioned modifications and onestep isolation methods to rationally tailor the aptamers of choice, a panel of aptamers with even greater affinity, avidity, specificity, selectivity and stability can be designed
Summary
Aptamers recognize and bind to their targets through 3-dimensional shapes and various physiochemical interactions, similar to antibody binding mechanisms. These interactions include, but are not limited to, hydrophobic, electrostatic, hydrogen bonding, van der Waals forces, base stacking, and shape complementarity. These functional nucleic acid molecules or aptamers owing to their flexibility, small size, and reduced steric hindrance can recognize biomolecules with ease, offering vast potential in diagnostics, therapeutics or drug-delivery systems (Catuogno et al, 2016; Sharma et al, 2016; Dhiman et al, 2017; Zhou and Rossi, 2017)
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