“An engineered SDL variant improves AppDNA-dependent single-stranded DNA ligation” (2026), by Fumihito Miura et al. Genetics and Biochemistry research paper. Original abstract below, unchanged. A free PDF is available.

An engineered SDL variant improves AppDNA-dependent single-stranded DNA ligation

Fumihito Miura, Yukiko Shibata, Miki Miura et al. · November 4, 2026 · Genetics and Biochemistry

Abstract

Efficient single-stranded DNA (ssDNA) ligation is required for library preparation from fragmented, chemically modified, and naturally occurring ssDNA. SDL is a recently identified ligase with strong ssDNA ligation activity, but conditions optimized for ATP-dependent ligation do not necessarily translate to reactions using pre-adenylated DNA donors (AppDNA). We first asked whether bypassing donor adenylation by supplying AppDNA could improve SDL-mediated ligation. Wild-type SDL rapidly ligated AppDNA in the absence of ATP, but product formation slowed after approximately 15 min and most AppDNA was converted to its 5′-phosphorylated form. Reasoning that a variant impaired in donor adenylation might also suppress this reverse conversion while retaining strand joining with AppDNA, we screened candidate substitutions and identified SDL-m5 (K239A). SDL-m5 retained efficient AppDNA ligation while markedly reducing AppDNA-to-pDNA conversion under the tested conditions. Wild-type SDL and SDL-m5 displayed distinct pH optima, and ligation efficiency across these conditions was inversely associated with sequence bias at the ligation junction. In cfDNA, replication-associated ssDNA, and bisulfite-converted DNA library workflows, SDL-m5 frequently yielded more library than wild-type SDL under AppDNA conditions. These results establish SDL-m5 as a practical variant for AppDNA-dependent ssDNA ligation and provide guidance for choosing ligation conditions in ssDNA library construction.

Free PDF DOI: 10.1038/s41598-026-79257-9