Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustering regularly spaced short palindromic repeats and the Cas system relies on primary spacer adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesirable off-target effects.
On September 18, 2024, the Su Xin team of Beijing University of Chemical Technology was inNature Biotechnology（IF=33）Published online entitled“Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allows PAM-independent targeting of double-stranded nucleic acids”The research paper,The study foundPhage lambda exonuclease and 5 '-phosphorylated DNA guidance allow PAM-independent double-stranded nucleic acid targeting.
Using single-molecule fluorescence resonance energy transfer analysis, the researchers discovered the enzymatic activity of bacteriophage lambda exonuclease (lambda Exo).It was found that 5 '-phosphorylated single-stranded DNA (pDNA) binds to complementary regions on dsDNA and DNA-RNA duplexes without the need for a PAM-like motif.After binding, the lambda Exo-pDNA system in Mg2 The presence catalyzes the digestion of pDNA into nucleotides. This process is very sensitive to mismatches within a wide range of pDNA binding regions, resulting in particular sequence specificity and reduced off-target effects in various applications. The absence of specific motifs, such as PAM sequences, greatly broadens the range of targets.The study demonstrates the lambda Exo-pDNA system as a versatile tool for molecular diagnostics, DNA computing, and gene imaging applications.
Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ site imaging. The clustered regularly interspaced short palindromic repeats (CRISPR) system has revolutionized the field of genome editing, expression interference, and molecular diagnostics.
Cas proteins mediate guide RNAs (gRNAs) that interrogate and cleave target nucleic acids by recognizing a protospacer adjacent motif (PAM). In addition, some Cas proteins exhibit indiscriminate cleavage activity on single-stranded reporter nucleic acids after target recognition. This feature makes it possible to design various advanced molecular diagnostic methods.The catalytically suppressed Cas protein (dead Cas, dCas) has been shown to be a powerful tool for identifying genes without cutting them. When dCas targets dsDNA, double-strand breaks are not induced, but rather binding to the target DNA is maintained. Thus, CRISPR-dCas can access highly localized genomic DNA without the need for global denaturation.
The researchers used CRISPR-dCas9 complexes in cell lines to achieve genomic site imaging.
However, the PAM-dependence and off-target effects of CRISPR-Cas systems not only limit their target profile, but also pose challenges for recognition specificity.
The bacteriophage lambda exonuclease (lambda Exo) recognizes 5 '-phosphorylated dsDNA and digests the 5'-phosphorylated strand in a procedural manner, releasing 5 'mononucleotides and leaving a long 3' overhang. Lambda Exo has low catalytic activity on non-phosphorylated DNA and single-stranded DNA(ssDNA). The crystal structure suggests that it forms a ring-shaped homotrimer with a funnel-shaped channel in the center for movement along the DNA. The dsDNA enters the wide end of the channel. The 5 'chain is digested at one of the three active sites, while the 3' chain passes through the narrow end of the channel and exits at the back. Its substrate binding and catalytic activity are largely influenced by key amino acid residues in its positively charged substrate pocket.
The typical properties of lambda Exo digestion of dsDNA 5 '-phosphorylated strands have been used to process phosphorylated amplification products, amplify signals, and detect mutations in ssDNA. However, the current study does not indicate that λExo can invade non-phosphorylated dsDNA or DNA-RNA double-stranded with the guide DNA strand.
Here, the authors discovered a nontrivial property of λExo using single-molecule fluorescence resonance energy transfer (smFRET) analysis. This property helps 5 '-phosphorylated ssDNA (pDNA) to bind and release dsDNA targets that have complementary sequences to pDNA.
in Ca2 pDNA and λExo were able to remain bound to the target in the presence of Mg2 In the presence of, pDNA is rapidly digested by lambda Exo. Absence of the target can completely prevent digestion of the pDNA.A single mismatch at or near the middle of the 5 'end of the pDNA-target complementary region prevented pDNA binding and cleavage, confirming that λExo activation is controllable. The advantage of this non-trivial nature has led to the potential for direct detection of synthetic dsDNA, DNA-RNA double-stranded and amplified products, using simple fluorescence quenching assays at physiological or room temperature.
The λExo-pDNA system was used to establish nucleic acid detection, which could rapidly and isothermally detect pathogen genes and related single nucleotide mutations. A DNA responsive circuit that can perform logic and amplification operations has been developed.
In addition, the lambda Exo-pDNA system was used to realize the human telomere genome site and the human mucin 4 gene (MUC4) in situ fluorescence imaging. This imaging method readily allows for signal enhancement using enzyme-free amplification.In addition, a single mismatch can be detected in locus imaging applications. In all of the above applications, the lambda Exo-pDNA complex does not require specific motifs, such as the PAM of the Cas protein, thereby greatly expanding the target spectrum. In addition, λExo-pDNA exhibits excellent single-mismatch sensitivity, allowing identification of a wide range of mutations.
Overall, the study reveals an important feature of λExo as a dsDNA recognition tool and reveals the enzymatic properties of λExo that enable sequence-specific identification of ds-nucleic acids at ambient temperatures.
Chongqing Chongfan Technology Co., Ltd. Optical platform film thickness meter OCT optical coherence tomography LabVIEW control
