Catalytic DNA‐Assisted Mass Production of Arbitrary Single‐Stranded DNA

Abstract Synthetic single‐stranded (ss) DNA is a cornerstone for life and materials science, yet the purity, quantity, length, and customizability of synthetic DNA are still limiting in various applications. Here, we present PECAN, p aired‐ e nd c utting a ssisted by D N Azymes (DNA enzymes or deoxyribozymes), which enables mass production of ssDNA of arbitrary sequence (up to 7000 nucleotides, or nt) with single‐base precision. At the core of PECAN technique are two newly identified classes of DNAzymes, each robustly self‐hydrolyzing with minimal sequence requirement up‐ or down‐stream of its cleavage site. Flanking the target ssDNA with a pair of such DNAzymes generates a precursor ssDNA amplifiable by pseudogene‐recombinant bacteriophage, which subsequently releases the target ssDNA in large quantities after efficient auto‐processing. PECAN produces ssDNA of virtually any terminal bases and compositions with >98.5 % purity at the milligram‐to‐gram scale. We demonstrate the feasibility of using PECAN ssDNA for RNA in situ detection, homology‐directed genome editing, and DNA‐based data storage.