Bacteriophage λ exonuclease and a 5'-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids.

Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustered regularly interspaced short palindromic repeats and Cas systems rely on protospacer-adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesired off-target effects. Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5'-phosphorylated single-stranded DNA (pDNA) to complementary regions on dsDNA and DNA-RNA duplexes, without the need for a PAM-like motif. Upon binding, the λExo-pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg 2+ . This process is sensitive to mismatches within a wide range of the pDNA-binding region, resulting in exceptional sequence specificity and reduced off-target effects in various applications. The absence of a requirement for a specific motif such as a PAM sequence greatly broadens the range of targets. We demonstrate that the λExo-pDNA system is a versatile tool for molecular diagnostics, DNA computing and gene imaging applications.