One-step selection of high-affinity COP1 aptamers

Aptamers are single-stranded DNA or RNA molecules that specifically bind to a wide range of target molecules with high affinity, making them powerful tools for synthetic biology. Traditional aptamer selection via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is labor-intensive and prone to nonspecific binding. To address these issues, we developed the Bead-based One-Step aptamer Selection (BOSS) method. As proof of concept, we used this method to target Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1 (AtCOP1), a conserved E3 ubiquitin ligase central to photomorphogenesis. We expressed the N-terminal RING (Really Interesting New Gene) domain of AtCOP1, immobilized it on Ni-NTA beads, and incubated the beads with a random ssDNA library. After washing the beads with washing buffer and replacing the buffer five times, followed by high-throughput sequencing, we identified the high-affinity aptamer Lib1-9, with a Kd of 11.14 nM for AtCOP1-RING. Lib1-9 demonstrated species specificity, showing strong binding to AtCOP1, but not to other COP1 homologs. Truncation analysis revealed that the core variable region (△LR) of Lib1-9 retained near-full binding affinity (Kd = 1.679 nM) to AtCOP1, which is comparable to the values observed for thrombin-binding aptamers. When we delivered Cy5-labeled aptamers into the hypocotyl cells of transgenic YFP-NLS-AtCOP1/cop1-4 A. thaliana plants, both Cy5-Lib1-9 and Cy5-Lib2-11 colocalized with YFP-COP1. The BOSS method is an efficient platform for plant aptamer development, enabling the rapid generation of tools for synthetic biology applications such as COP1 biosensing and optogenetic control.