一种非荧光共振能量转移(FRET)DNA报告分子,在核酸酶消化后会改变荧光颜色。
A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.
作者信息
Hong Soonwoo, Walker Jada N, Luong Aaron T, Mathews Jonathan, Shields Samuel W J, Kuo Yu-An, Chen Yuan-I, Nguyen Trung Duc, He Yujie, Nguyen Anh-Thu, Ghimire Madhav L, Kim Min Jun, Brodbelt Jennifer S, Yeh Hsin-Chih
机构信息
Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA.
Department of Chemistry, The University of Texas at Austin, Austin, TX, USA.
出版信息
Nat Nanotechnol. 2024 Jun;19(6):810-817. doi: 10.1038/s41565-024-01612-6. Epub 2024 Feb 13.
Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion. Notably, a 90 nm red shift in emission is observed upon reporter cleavage, a result unattainable by a simple donor-quencher FRET reporter. Electrospray ionization-mass spectrometry results suggest that the stoichiometric change of the silver nanoclusters from Ag (in the intact DNA host) to Ag (in the fragments) is probably responsible for the emission colour change observed after reporter digestion. Our results demonstrate that DNA-templated silver nanocluster probes can be versatile reporters for detecting nuclease activities and provide insights into the interactions between nucleases and metallo-DNA nanomaterials.
荧光共振能量转移(FRET)报告分子通常用于核酸扩增检测的最后阶段,以指示核酸靶标的存在,在该阶段,荧光通过切割FRET报告分子的核酸酶得以恢复。然而,制造过程中对双重标记和纯化的需求导致FRET报告分子成本高昂。在此,我们展示了一种低成本的银纳米簇报告分子,其开/关切换机制不依赖FRET,而是基于一种簇转化过程,该过程在核酸酶消化后导致荧光颜色变化。值得注意的是,报告分子切割后观察到发射峰有90纳米的红移,这是简单的供体-猝灭剂FRET报告分子无法实现的结果。电喷雾电离质谱结果表明,银纳米簇从完整DNA宿主中的Ag到片段中的Ag的化学计量变化可能是报告分子消化后观察到的发射颜色变化的原因。我们的结果表明,DNA模板化的银纳米簇探针可以作为检测核酸酶活性的通用报告分子,并为核酸酶与金属DNA纳米材料之间的相互作用提供见解。
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