噬菌体λ外切酶和5′-磷酸化DNA向导可实现原间隔相邻基序（PAM）非依赖的双链核酸靶向，这一成果由北京化工大学苏昕教授研究组经过不懈努力而取得。相关论文发表在2024年9月18日出版的《自然—生物技术》杂志上。

利用单分子荧光共振能量转移分析，研究人员揭示了噬菌体λ外切酶（λExo）的酶活性。研究展示了5′-磷酸化单链DNA（pDNA）与dsDNA以及DNA-RNA双链互补区的结合，而不需要类似PAM的基团。结合后，λExo-pDNA系统会在Mg²⁺的催化下消化pDNA成为单核苷酸。

这一过程对pDNA结合区内的错配非常敏感，因此在各种应用中都具有极高的序列特异性，减少了脱靶效应。由于不需要PAM序列等特定结构，因此大大拓宽了靶标范围。该研究表明λExo-pDNA系统是可用于分子诊断、DNA计算和基因成像的多功能工具。

据悉，双链核酸的序列特异性识别对于分子诊断和原位成像至关重要。簇状规则间隔短回文重复序列和Cas系统依赖于PAM的双链DNA（dsDNA）识别，这限制了可靶序列的范围，并导致脱靶效应。

附：英文原文

Title: Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids

Author: Fu, Shengnan, Li, Junjie, Chen, Jing, Zhang, Linghao, Liu, Jiajia, Liu, Huiyu, Su, Xin

Issue&Volume: 2024-09-18

Abstract: 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 Mg2+. 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.

DOI: 10.1038/s41587-024-02388-9

Source: https://www.nature.com/articles/s41587-024-02388-9