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

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, paired-end cutting assisted by DNAzymes (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.