A Self-Cleaving DNAzyme Adapter as a Cost-Effective Alternative to USER-Cleaving Adapters for Illumina Sequencing

In Illumina next-generation sequencing, target DNA fragments are ligated to adapters containing chemically modified uracil bases, which are subsequently cleaved by uracil-specific excision reagent (USER) to generate the Y-shaped structure essential for downstream sequencing. Herein, we employed the trans-cleaving F-8 DNAzyme as a starting scaffold (DNAzyme reagent) and performed site-directed mutagenesis within its catalytic core. By combining highly active mutations, we identified the variant F-85A6A, which exhibited the highest cleavage yield and demonstrated superior adapter-cleavage efficiency relative to USER, underscoring its potential as a cost-effective USER substitute. We then integrated the catalytic core of the F-8 DNAzyme into the Illumina adapter architecture to design F-8H1. Sequential optimization of the binding arm length and further mutagenesis yielded the highly active mutant H6-13A42A. The 5′ end of H6-13A42A was modified with a phosphate group to construct a complete self-cleaving adapter. In a simulated dual-adapter sequencing structure, the self-cleaving adapter achieved cleavage efficiency comparable to that of USER-treated Illumina adapters while reducing reagent costs by 97.5%, thereby offering an economical molecular tool for advancing DNA sequencing technology.