Sequence-Specific Recognition of Double-Stranded Nucleic Acids by λ Exonuclease-Phosphorylated DNA Mediated Target Interrogation

Abstract The CRISPR-Cas system, guided by an RNA molecule, cleaves double-stranded DNA targets in proximity to a protospacer adjacent motif (PAM), enabling the applications in genetic editing/interference and molecular diagnostics1–3. However, the reliance on PAM-dependent recognition and cleavage has limited the range of targetable sequences and led to undesired off-target effects, resulting in false activations4–7. Here, we conducted single-molecule FRET (smFRET) analysis and discovered a remarkable enzyme activity of λ Exonuclease (λ Exo)8. This activity involves the binding of 5’-phosphorylated single-stranded DNA (pDNA) to double-stranded (ds-) DNA and DNA-RNA duplexes, without the need for a specific PAM-like motif. Upon binding to its complementary region on nucleic acid targets, the λ Exo-pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg2+. Importantly, this process is sensitive to mismatches, resulting in exceptional sequence specificity and greatly reduced off-target effects. Leveraging this unique property, we successfully demonstrated various applications, such as molecular diagnostics, synthetic DNA circuits, and in situ genomic imaging. Direct recognition of ds- nucleic acid was achieved by simple fluorescence reporting. DNA circuits9,10 with ds- nucleic acid inputs were established to execute the logical operation and signal amplification. The λ Exo-pDNA system enabled in situ imaging of genomic loci in fixed cells, directly observing both high-copy loci (e.g., telomeres) and low-copy loci (e.g., MUC-4). The non-trivial activity of λ Exo has unlocked PAM-independent target recognition and improved sequence specificity and accuracy. This discovery holds immense promise for advancing gene regulation and molecular diagnostics in the future.