Jie Li, Ying Yao, Shuning Zhang, Wei Wang, Wanting Bi, Jian Lv, Wei Hou, Peixiang Ma, Hongtao Xu

Chem

DOI:10.1016/j.chempr.2026.102945

Abstract

To address the challenges associated with the construction and selection of single-stranded DNA-encoded libraries (ssDELs), an efficient and precise short-splint RNA-mediated single-stranded DNA (ssDNA) ligation system has been developed. This system exhibits superior discrimination of single-base mismatches at oligonucleotide ligation junctions and requires only 12–14 nucleotides to achieve optimal ligation. By leveraging these advantages, we have established a concise ssDEL-encoding platform, as evidenced by the efficient synthesis and selection of pilot libraries ranging from 10⁴ to 2.95 × 10⁶ members. Additionally, capitalizing on the high sensitivity of this ligation system, a SplintR ligase-mediated proximity ligation (SIMPL)-based polymerase chain reaction (PCR) method has been developed. This method achieves remarkable sensitivity, enabling the linking of chemical and phenotypic information at detection limits as low as zeptomole levels. This capability facilitates significant hit enrichment ratios and enables the identification of a novel carbonic anhydrase XII (CAXII) binder with picomolar affinity through label-free, live-cell-based ssDEL selection.