The electrochemical aptamer-based (E-AB) sensing platform appears to be a convenient (rapid, single-step, and calibration-free) and modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living *

Artificial receptors for hydrophobic molecules usually have moderate affinities and limited selectivities. We describe three new classes of high affinity hydrophobic receptors for nonaromatic steroids based on deoxyribonucleotides, obtained through five high stringency selections coupled with tailored counter-selections. The isolation of multiple classes of high affinity steroid receptors demonstrates the surprising breadth of moderately sized hydrophobic binding motifs (<40 nucleotides) available to natural nucleic acids. Studies of interactions with analogs indicate that two classes, four-way junctions and 4XG motifs, comprise receptors with shapes that prevent binding of specific steroid conjugates used in counter-selections. Furthermore, they strongly prefer nonhydroxylated steroid cores, which is typical for hydrophobic receptors. The third new class accommodates hydroxyl groups in high-affinity, high-selectivity binding pockets, thus reversing the preferences of the first two classes. The high-affinity binding of aptamers to targets efficiently inhibits double-helix formation in the presence of the complementary oligonucleotides. The high affinity of some of these receptors and tailored elimination of binding through counter-selections ensures that these new aptamers will enable clinical chemistry applications.

Aptameric receptors are important biosensor components, yet our ability to identify them depends on the target structures. We analyzed the contributions of individual functional groups on small molecules to binding within 27 target-aptamer pairs, identifying potential hindrances to receptor isolation—for example, negative cooperativity between sterically hindered functional groups. To increase the probability of aptamer isolation for important targets, such as leucine and voriconazole, for which multiple previous selection attempts failed, we designed tailored strategies focused on overcoming individual structural barriers to successful selections. This approach enables us to move beyond standardized protocols into functional group–guided searches, relying on sequences common to receptors for targets and their analogs to serve as anchors in regions of vast oligonucleotide spaces wherein useful reagents are likely to be found.