High-Affinity Nucleic-Acid-Based Receptors for Steroids

Abstract

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.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates

“…51 Even so, chemical intuition, carefully designed nucleic acid libraries, and stringent counter-selection protocols can lead to aptamers that distinguish physiologically important yet structurally similar small molecules. 20,21,52–54 Multiplexed small-molecule-tethered substrates that enable relative comparisons of dissociation constants for aptamer binding to specific vs . nonspecific or closely structured counter-targets reduces measurement variabilities across different substrates.…”

Engineering Aptamer Switches for Multifunctional Stimulus‐Responsive Nanosystems

“…Rapid progress is now being made in this area—for example, the Stojanovic group has devised a strand‐displacement SELEX method to develop aptamer switches to steroids, neurotransmitters, and other small molecules of interest. [ 94 ] We have also highlighted here our own work with pH‐responsive aptamer screening, and believe that there are abundant opportunities to identify additional selection strategies for the routine isolation of DNA molecules that undergo precise switching in response to a variety of biological and other triggers in complex environments. Non‐natural xeno nucleic acids (XNA), which incorporate additional chemical modifications to the nucleotide backbone or nucleobase, could also offer a valuable opportunity for extending the functionality of aptamer switches.…”

Single-molecule DNA origami aptasensors for real-time biomarker detection

“…Aptamers are well-placed to be implemented as sensors, both in the bulk and at the single-molecule level, due to their high affinity and specificity for analytes as well as their facile synthesis. 1,3,4,7,8,27,28 Moreover, there are numerous examples of single-molecule detection in electrical, [10][11][12][13][14] fluorescent, [15][16][17][18] and mechanical systems, 19 as well as in atomic force microscopy (AFM)-based analysis. [20][21][22][23] However, previously reported aptamer-based single-molecule sensors are typically based on aptamers with well-documented conformations or have not been observed in real-time, so the scope of applications has been limited.…”

“…18,[20][21][22][23][42][43][44] Herein, we explore the use of DNA nanostructures as platforms to monitor aptamers inherent conformational changes upon analyte binding, with single-molecule resolution and realtime capability. In particular, we designed a DNA origami with a single cortisol-sensing duplexed aptasensor on one face, 27 which could be immobilised to surfaces via molecular anchors on the opposite face. Using this approach, we could be certain that a single aptamer would be present within the range of the origami, while protruding from the surface for adequate interaction with the environment.…”