Selection of aptamers using β-1,3-glucan recognition protein-tagged proteins and curdlan beads

Abstract

RNA aptamersare nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilize target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins but also to the protein tags and matrix beads. In this study, we performed SELEX using β-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilize the acute myeloid leukaemia 1 (AML1) Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Keywords: SELEX.Abbreviations: AML1, acute myeloid leukaemia 1; βGRP, β-1,3-glucan recognition protein; GST, glutathione S-transferase; His-tag, poly histidine tag; HTS, high-throughput sequencing; MBP, maltose-binding protein; RD, Runt domain; RUNX1, RUNX family transcription factor 1; SELEX, systematic evolution of ligands by exponential enrichment; SPR, surface plasmon resonance; aptamer; curdlan; βGRP.