Fu Shengnan, Li Junjie, Chen Jing, Zhang Linghao, Liu Jiajia, Liu Huiyu, Su Xin
Beijing Advanced Innovation Center for Soft Matter Science and Engineering and State Key Laboratory of Organic-Inorganic Composites, Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
Nat Biotechnol. 2024 Sep 18. doi: 10.1038/s41587-024-02388-9.
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. 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.
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。成簇规律间隔短回文重复序列（CRISPR）及其相关蛋白（Cas）系统依赖于原间隔序列临近基序（PAM）依赖性双链DNA（dsDNA）识别，这限制了可靶向序列的范围并导致不期望的脱靶效应。通过单分子荧光共振能量转移分析，我们发现了噬菌体λ核酸外切酶（λExo）的酶活性。我们展示了5'-磷酸化单链DNA（pDNA）与dsDNA和DNA-RNA双链体上互补区域的结合，而无需类似PAM的基序。结合后，λExo-pDNA系统在镁存在的情况下将pDNA催化消化为核苷酸。这一过程对pDNA结合区域内广泛范围内的错配敏感，在各种应用中产生了卓越的序列特异性并减少了脱靶效应。对诸如PAM序列等特定基序的需求缺失极大地拓宽了靶标的范围。我们证明λExo-pDNA系统是用于分子诊断、DNA计算和基因成像应用的通用工具。
