Direct Selection Strategy for Isolating Aptamers with pH-Sensitive Binding Activity.

Chelsea K L Gordon,Hyongsok Tom Soh,Michael Eisenstein

doi:10.1021/acssensors.8b00945

Chelsea K L Gordon, Hyongsok Tom Soh + Show 1 more

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https://doi.org/10.1021/acssensors.8b00945

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Journal: ACS Sensors	Publication Date: Dec 6, 2018
Citations: 20	License type: cc-by

Affiliation: Stanford University, CZ Biohub

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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.

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C ellular pH is carefully regulated, as it plays an essential role in many critical functions including energy generation and maintenance of protein structure and function.[1−3] differences in pH help to control the binding and release of important biomolecules by pHregulated receptors
We have designed a selection procedure that enables us to isolate aptamers that exhibit different target affinities at pH 7.4 compared to pH 5.2, which we demonstrate by generating pHsensitive aptamers for streptavidin
We describe a rapid and high-throughput method that enables us to screen for pH-sensitive derivatives of existing aptamers based on particle display, without the need for laborintensive aptamer engineering procedures.[10,26]

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Introduction

C ellular pH is carefully regulated, as it plays an essential role in many critical functions including energy generation and maintenance of protein structure and function.[1−3] differences in pH help to control the binding and release of important biomolecules by pHregulated receptors. We have designed a selection procedure that enables us to isolate aptamers that exhibit different target affinities at pH 7.4 compared to pH 5.2, which we demonstrate by generating pHsensitive aptamers for streptavidin. Our procedure (Figure 1B) is a variation on the previously described particle display platform, a high-throughput aptamer screening strategy based on FACS that enables the analysis of individual aptamer binding characteristics at a rate of ∼106 sequences/h.19 The critical difference between this platform and conventional SELEX

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