Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol

Abstract

The first-known aptamer for the stress biomarker cortisol was selected using a tunable stringency magnetic bead selection strategy. The capture DNA probe immobilized on the beads was systematically lengthened to increase the number of bases bound to the complementary pool primer regions following selection enrichment. This resulted in a single sequence (15-1) dominating the final round 15 pool, where the same sequence was the second-highest copy number candidate in the enriched pool with the shorter capture DNA probe (round 13). A thorough analysis of the next-generation sequencing results showed that a high copy number may only correlate with enhanced affinity under certain stringency and enrichment conditions, in contrast with prior published reports. Aptamer 15-1 demonstrated enhanced binding to cortisol (K(d) = 6.9 ± 2.8 μM by equilibrium dialysis; 16.1 ± 0.6 μM by microscale thermophoresis) when compared with the top sequence from round 13 and the negative control progesterone. Whereas most aptamer selections terminate at the selection round demonstrating the highest enrichment, this work shows that extending the selection with higher stringency conditions leads to lower amounts eluted by the target but higher copy numbers of a sequence with enhanced binding. The structure-switching aptamer was applied to a gold nanoparticle assay in buffer and was shown to discriminate between cortisol and two other stress biomarkers, norepinephrine and epinephrine, and a structurally analogous biomarker of liver dysfunction, cholic acid. We believe this approach enhances aptamer selection and serves as proof-of-principle work toward development of point-of-care diagnostics for medical, combat, or bioterrorism targets.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

“…This result shows that copy number from a sequenced pool and affinity for the target may only be associated under certain conditions 24,34 , in contrast with a prior report implying a direct correlation 35 . It also highlights the benefit of monitoring enrichment by NGS, rather than solely by the % eluted strategy common in most selections, which may vary naturally from one round to the next as higher stringency conditions are applied.…”

“…These higher sequence reads present a greater coverage of the total number of sequences in the pool, providing a more complete picture of the diversity of sequences. NGS also removes the cloning bias 32 , and allows for sequencing of multiple pools in a single batch with the addition of bar codes 24,33 . NGS was used to analyze the progression of enrichment from three separate pools in this sample selection (Figure 2).…”

“…Previous studies examined the binding of these two sequences by microscale thermophoresis and equilibrium dialysis 24 . The results showed significant cortisol binding for sequence 15-1 (K d = 6.9 ± 2.8 µM by equilibrium dialysis; 16.1 ± 0.6 µM by microscale thermophoresis) while minimal binding was observed between cortisol and sequence 15-3.…”

“…Therefore, % eluted can serve as a guide for adjusting the stringency during the course Sequence 15-1 was applied to an AuNP assay to determine if a sequence selected in a manner to produce a structure-switching aptamer would function in an assay that relies on a structural change following target binding to produce a response. Previous initial studies showed that the AuNP assay with 15-1 responded to the stress biomarker cortisol, but not to two other markers of stress, epinephrine and norepinephrine, or cholic acid, a marker of liver disease structurally similar to cortisol 24 . The response was observed in the normal range of free cortisol reported in human serum (~150-600 nM) 6 , validating that the aptamer selected for a structural change upon cortisol binding produces a response in the AuNP assay.…”

“…This protocol is in contrast with the radioligand enrichment detection scheme used in legacy methods 17 . The proposed method also incorporated an increase in the length of the cDNA capture probes used in the selection to increase the partitioning efficiency and effectively tune the stringency of the selection after maximal enrichment was observed 24 . The tuned stringency lead to a lower total percentage of DNA eluted by the target in the final pool, but resulted in higher copy numbers of a sequence that bound with enhanced affinity compared to the highest copy number sequence from an earlier round.…”