Princess Margaret Cancer Centre and Techna Institute, University Health Network, 101 College St., Toronto, ON, Canada.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Angew Chem Int Ed Engl. 2017 Oct 23;56(44):13704-13708. doi: 10.1002/anie.201707366. Epub 2017 Sep 27.
MicroRNA detection is a valuable method for determining cell identity. Molecular beacons are elegant sensors that can transform intracellular microRNA concentration into a fluorescence intensity. While target binding enhances beacon fluorescence, the degree of enhancement is insufficient for demanding applications. The addition of specialty nucleases can enable target recycling and signal amplification, but this process complicates the assay. We have developed and characterized a class of beacons that are susceptible to the endogenous nuclease Argonaute-2 (Ago2). After purification of the complex by co-immunoprecipitation, microRNA:Ago2 cleavage (miRACle) beacons undergo site- and sequence-specific cleavage, and show a 13-fold fluorescence enhancement over traditional beacons. The system can be adapted to any microRNA sequence, and can cleave nuclease-resistant, non-RNA bases, potentially allowing miRACle beacons to be designed for cells without interference from non-specific nucleases.
微 RNA 检测是确定细胞身份的一种有价值的方法。分子信标是一种优雅的传感器，可以将细胞内微 RNA 浓度转化为荧光强度。虽然靶标结合增强了信标荧光，但增强程度不足以满足高要求的应用。添加特殊的核酸酶可以实现靶标循环和信号放大，但这一过程使检测复杂化。我们开发并表征了一类对内源性核酸酶 Argonaute-2 (Ago2)敏感的信标。通过共免疫沉淀法纯化复合物后，微 RNA:Ago2 切割 (miRACle) 信标发生位点和序列特异性切割，与传统信标相比，荧光增强 13 倍。该系统可以适应任何微 RNA 序列，并且可以切割核酸酶抗性、非 RNA 碱基，这可能使 miRACle 信标能够设计用于不受非特异性核酸酶干扰的细胞。
