A direct selection strategy for isolating aptamers with pH-sensitive binding activity

Abstract

An aptamer reagent that can switch its binding affinity in a pH-responsive manner would be highly valuable for many biomedical applications including imaging and drug delivery.Unfortunately, the discovery of such aptamers is difficult and only a few have been reported to date. Here we report the first experimental strategy for generating pH-responsive aptamers through direct selection. As an exemplar, we report streptavidin-binding aptamers that retain nanomolar affinity at pH 7.4 but exhibit a ~100-fold decrease in affinity at pH 5.2. These aptamers were generated by incorporating a known streptavidin-binding DNA motif into an aptamer library and performing FACS-based screening at multiple pH conditions. Upon structural analysis, we found that one aptamer's affinity-switching behavior is driven by a noncanonical G-A base-pair that controls its folding in a highly pH-dependent manner. We believe our strategy could be readily extended to other aptamer-target systems because it does not require a priori structural knowledge of the aptamer or the target.

A Wireless, Regeneratable Cocaine Sensing Scheme Enabled by Allosteric Regulation of pH Sensitive Aptamers

“…Figure S6 shows the regeneration performance of sensors functionalized with pH sensitive antistreptavidin and antithrombin aptamers and the underlying mechanism based on the allosteric regulation of the DNA sequences: at a neutral pH condition, both aptamers are in a folded conformation for the binding of streptavidin and thrombin molecules with a high affinity. 50,51 Following a very similar working principle to that of the anti-cocaine aptamers, the addition of extra protons in an acidic environment results in a mismatch between the base pair G and A, leading to a disruption of the original conformations and, accordingly, the release of adsorbed analytes for surface regeneration. In both cases, the devices can fully restore the sensitivity after an acid treatment (pH = 5.0) for 60 min (for streptavidin: −3.83 ± 0.78 mV/dec (before) vs −4.44 ± 0.43 mV/dec (after); for thrombin: −3.34 ± 0.91 mV/dec (before) vs −3.42 ± 0.72 mV/dec (after)).…”

“…Thiolated anti-cocaine aptamers (with pH sensitive motif: 5′-CCC TCT ATT TCT CTC CCT TTG GGA GAC AAG GAA AAT CCT TCA ATG AAG TGG GTC GAC A/3Thio-MC3-D/-3′; without pH sensitive motif: 5′-GGG AGA CAA GGA AAA TCC TTC AAT GAA GTG GGT CGA CA/3Thio-MC3-D/-3′), thiolated antithrombin aptamers (5′-GGT TGG TGT GGT TGG CTC TAA AAA AAA AAA AAA A/3Thio-MC3-D/-3′), and thiolated antistreptavidin aptamers (5′-ATA CCA GCT TAT TCA ATT ATT GAC CGC TGT GTG ACG CAA CAC TCA ATT CTT GGA TCT CGC TGC ACA CAG ATA GTA AGT GCA ATC T/3Thio-MC3-D/-3′) were synthesized by Integrated DNA Technologies based on sequences reported in previous studies. 29,50,51 Dissolving as-purchased aptamers in 1× TE solution with 10 mM DTT reduced the disulfide bonds and yielded a solution of aptamers with −SH groups at the 3′ end (concentration: 100 μM). Centrifuging the resulting solution in a MySpin12 (Thermo Fisher Scientific) at 2038 relative centrifugal force (RCF) (2600 rpm) for 4 min removed additional DTT.…”

Selection of Peptides that Associate with Dye-Conjugated Solid Surfaces in a pH-Dependent Manner Using cDNA Display

“…25 Moreover, immobilized azobenzene molecules seem to yield more mixed cis/trans isomers at the photostationary state. 25 Although a few DNA aptamers that show pH-dependent binding to the targets have been developed recently, 26,27 there is no report of the selection of pH-dependent peptide aptamers.…”

Directed Evolution of Aptamer Discovery Technologies

“…Antibodies are one of the foundational tools of molecular biology and biomedical research, but nucleic acid-based aptamers offer some critical advantages relative to protein-based affinity reagents. Aptamers are readily chemically synthesized in a reproducible manner and can be engineered to incorporate useful functionalities such as structure switching 1 and selective response to environmental conditions such as pH 2 or redox states 3 . Furthermore, aptamers can be generated for low-molecular-weight targets that would be challenging for antibody generation, such as small-molecule ligands 4,5 and even metal ions 6 .…”