introduction
The specific recognition of double-stranded nucleic acids is a core technology in the field of life sciences. The sequence limitations and off-target effects of current double-stranded nucleic acid recognition methods have long plagued key biomedical technologies such as molecular diagnosis and pathological imaging. At present, it mainly relies on CRISPR and its nuclease system discovered by United States scientists. CRISPR systems have transformed genome editing, expression interference, and molecular diagnostics [1]. CRISPR-associated proteins (Cas) cleave target nucleic acids by recognizing adjacent motifs (PAMs) of the original spacer under the guidance of crRNAs [2]. However, the PAM-dependent and off-target effects of the CRISPR-Cas system limit the range of their targets and affect their biosafety. Although scholars around the world are constantly improving CRISPR and its nucleases, there are no new technologies that have completely solved this problem.
2024年9月18日,北京化工大学苏昕教授和刘惠玉教授课题组在Nature Biotechnology发表了题为Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids的研究论文以及Targeting double-stranded nucleic acids using the λ Exo-pDNA system的研究简报,报道了一种来自噬菌体λ外切酶(λ Exo)的新特性,它可以在引导DNA的帮助下特异性靶向双链核酸序列,解决了现有技术的序列限制性与脱靶效应。
The research team demonstrated the mechanism by which λ Exo can bind to double-stranded DNA (dsDNA) or DNA-RNA duplex containing pDNA complementary regions under the guidance of 5'-phosphorylated single-stranded DNA (pDNA) under the guidance of 5'-phosphorylated single-stranded DNA (pDNA). This binding can be achieved at room temperature or body temperature and does not require any specific motifs such as PAM. After binding, λ Exo digests pDNA into nucleotides in the presence of Mg2+. With this feature, the λ Exo-pDNA System can detect double-stranded genes and single nucleotide mutations at room temperature and body temperature, as well as perform logic operations and signal amplification. λ Exo-pDNA can also be used for in situ fluorescence imaging of genomic loci.
模式图(Credit: Nature Biotechnology)
In conclusion, the λ Exo-pDNA system has significant improvements over existing tools such as TALEN, PfAgo, and CRISPR-Cas in terms of target range, room temperature manipulation, and sequence specificity. The λ Exo-pDNA system has the potential to become a next-generation tool in areas such as molecular diagnostics, DNA computation, and in situ imaging.
bibliography
1.Swarts, D.C. et al. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic. Acids. Res. 43, 5120-5129 (2015).
2.Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347-355 (2014).
https://www.nature.com/articles/s41587-024-02388-9
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