Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

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.

Keywords: Kd; SELEX; affinity; aptamers; limit of detection; selectivity; sensitivity; specificity.

Figure 1

Illustration of a cancer biomarker (ERK2) aptamer pull-down assay on aptamer-coated magnetic beads (MBs) performed in serum and analyzed by gel electrophoresis and mass spectrometry. The “clean” ~43.5 kD ERK2 band shown in the gel and clear mass spectral signature virtually free of interfering signals from serum were likely due to original aptamer selection in diluted serum as well as the addition of 2 mM EDTA to chelate free calcium ions, thereby enhancing aptamer specificity (Bruno, 2017b). Anti-ERK2 aptamers are shown as wavy red lines attached to MBs either covalently or via streptavidin-biotin linkages which then capture any free extant ERK2 or spiked recombinant ERK2 (rERK2) in serum samples. Following MB collection with a strong permanent magnet (pull-down), removal of serum, several buffer washes and elution of the ERK2 from the aptamer MBs in strong (0.1 M) HCl, followed by removal of the aptamer-MBs and neutralization of ERK2 in the acid with 0.1 M NaOH, the resultant eluate is electrophoresed in polyacrylamide and analyzed by mass spectrometry to validate its molecular weight (MW) and probable identity. A real experimental Coomassie Blue-stained 12% polyacrylamide gel and mass spectrum are shown from experimental pull-down assay runs.

Figure 2

Comparison of relative sensitivities, turnaround time and cost of various K_d estimation techniques. K_d, Apparent dissociation constant (Molar); UV-Vis, Ultraviolet-Visible; ALISA, Aptamer-linked immobilized sorbent assay; ELONA, Enzyme-linked oligonucleotide sorbent assay; ELASA, Enzyme-linked aptamer sorbent assay.

Figure 3

Utility of aptamer cocktail in enhancing signal detection. Schematic concept of signal enhancement by aptamer cocktails for microbial cell detection and increase in fluorescence intensity of bacterial cell suspensions by the interaction with single aptamers or aptamer cocktails.

Figure 4

Identification of two candidate aptamers which were selected to bind Listeria monocytogenes specific peptide epitope (QQQTAPKAPTE). Top—initial ELISA-like (ELASA) screening assay results, which identified p60 29R and 34F as the best or highest affinity aptamers for binding to the peptide. Bottom—ELASA results for these two aptamers vs. a variety of Listeria species. Both aptamers exhibited a preference for binding to immobilized L. monocyogenes whole viable cells. Bar heights and error bars represent the Means ± 2 Standard Deviations of six independent ELASA readings (Bruno and Sivils, 2017).

Figure 5

Results of initial YASARA-based 3-D molecular modeling to identify where the PSA variant isoleucine (I) vs. threonine (T) peptide regions bound to the candidate aptamer. Top—trace models of the aptamer docked with the two variant PSA peptides reveal binding to the same loop structure. Bottom−3-D space-filling ball models of the same binding events. Peptides are encircled in each case to distinguish their locations within the loop structure designated Loop 1 in the docked aptamer-ligand complexes.

Figure 6

Close up views of the top PSA aptamer binding to both, the I and T peptide variants. This tube structure docking analysis revealed the presence of a common proximal adenine in the aptamer binding site as indicated by arrows directly across from the indicated isoleucine (I) or threonine (T), which could be replaced by a diaminopurine (DAP) base to alter binding in the region and potentially discriminate the I- vs. T-PSA peptide variants.

Figure 7

ELASA results demonstrating differential binding of the DAP-modified aptamer to the PSA variant peptides. The DAP-modified aptamer demonstrated ~ 20% greater affinity for the PSA-isoleucine (I) vs. the PSA-threonine (T) variant. Bar heights and error bars represent the Means ± 2 Standard Deviations of four independent enzyme-linked (ELASA) absorbance at 405 nm.