https://doi.org/10.1002/anie.202510518

Stangherlin, Stefen; Malloch, Tyler; Clarke, Anthony J.; Liu, Juewen (2025) Green Fluorescent Protein SELEX: Immobilization Chemistry and His-Tag Epitope Bias, Angewandte Chemie International Edition, https://doi.org/10.1002/anie.202510518

Despite numerous DNA aptamers for proteins having been reported, a model system allowing the use of cost-effective proteins, unmodified DNA, and convenient homogeneous assays is still lacking, which has in turn limited not only fundamental studies of aptamers but also their translation to practical applications. Herein, three separate green fluorescent protein (GFP) selections were carried out using both non-tagged and His-tagged GFP immobilized on either NHS-activated resin or Co2+ affinity resin. Only the GFP/NHS system resulted in aptamers that consistently bind to unmodified GFP, whereas the His-tagged GFP yielded aptamers biased toward the His-tag epitope. Sequence alignment and fluorescence polarization assays indicate that many previously published aptamers bound to the His-tag instead of the intended protein. This work not only obtained a model aptamer for proteins but also revealed critical information on bias toward His-tags during aptamer selections.

https://doi.org/10.1002/anie.202510518