The specific recognition of double-stranded nucleic acids is a core technology in the field of life sciences, however, the sequence limitations of this method and off-target effects have long plagued key biomedical technologies such as molecular diagnostics and pathological imaging. Although CRISPR and its nucleic acid system discovered by American scientists are now widely used, its dependence on specific motifs and off-target effects not only limit its scope of application, but also affect its biosafety. Currently, scholars in various countries are in a fierce race to improve CRISPR and its nuclease, but no new technology has yet to completely solve this problem.
On September 18, 2024, Professor Su Xin's group published a paper entitled “Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleases” in Nature Biotechnology. independent targeting of double-stranded nucleic acids” and ‘Targeting double-stranded nucleic acids using the λ Exo-pDNA system’ in Nature Biotechnology. -pDNA system”, reported a new property from phage λ exonuclease (λ Exo), which can specifically target double-stranded nucleic acid sequences with the help of guide DNA, solving the sequence restriction and off-target effect of the existing technology.
Figure Schematic diagram of λ Exo targeting double-stranded nucleic acids and cleaving pDNA; the novel properties of λ Exo are applied to molecular diagnostics, DNA circuits, and in situ genetic imaging.
In this paper, we demonstrate the mechanism by which λ Exo, guided by 5'-phosphorylated single-stranded DNA (pDNA), can bind to double-stranded DNA (dsDNA) or DNA-RNA duplexes containing complementary regions of pDNA by single-molecule FRET (smFRET) analysis. This binding interaction can be achieved at room or body temperature and does not require any specific motifs such as PAM-like. Upon binding, λ Exo digests pDNA into nucleotides in the presence of Mg2+. Using this property, the λ Exo-pDNA system is able to detect double-stranded genes and single-nucleotide mutations at room and body temperatures, as well as perform logic operations and signal amplification. λ Exo-pDNA can also be used for in-situ fluorescence imaging of genomic loci. λ Exo-pDNA system is more efficient in terms of target range, ambient operation, and sequence specificity than existing tools such as TALEN, PfAgo, and CRISIS, and it can be used for in situ fluorescence imaging of genomic loci. The λ Exo-pDNA system may become a next-generation tool for molecular diagnostics, DNA computation and in situ imaging.
The first author of the paper is Shengnan Fu, a PhD student at the School of Life Sciences and Technology, and the corresponding author is Prof. Xin Su at the School of Life Sciences and Technology. Beijing University of Chemical Technology is the sole author of the paper.
Prof. Xin Su's profile:
Prof. Su Xin is a professor and doctoral supervisor of Beijing University of Chemical Technology (BUCT), and a national young talent.He received his PhD degree from BUCT in 2015, and has been an exchange student at the University of Michigan, U.S.A. He has been a professor and doctoral supervisor of BUCT since 2021. His main research interests include in vitro diagnostics, biosensing, and bio-nanotechnology. He has presided over 1 national talent program, 3 National Natural Science Foundation of China (NSFC) projects, 2 national key R&D projects, and 1 Beijing Natural Science Foundation. He has published more than 50 papers in Nature Biotechnology (2), ACS Nano, Nano Letters (2), Adv Func Mater, Nano Today as the first or corresponding author, and has been awarded 5 national invention patents. He serves as an expert committee member of the Chinese Society of Integrative Medicine and Western Medicine, and is a young editorial board member of Exploration journal. He has been honored with the Beijing Outstanding Talent and the Zhu Liangyi Award of the Chinese Society of Instrumentation and Instrumentation.
