Green Christopher M, Spangler Joseph, Susumu Kimihiro, Stenger David A, Medintz Igor L, Díaz Sebastián A
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, D.C.20375, United States.
Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, D.C.20375, United States.
ACS Nano. 2022 Dec 27;16(12):20693-20704. doi: 10.1021/acsnano.2c07749. Epub 2022 Nov 15.
Strategies utilizing the CRISPR/Cas nucleases Cas13 and Cas12 have shown great promise in the development of highly sensitive and rapid diagnostic assays for the detection of pathogenic nucleic acids. The most common approaches utilizing fluorophore-quencher molecular beacons require strand amplification strategies or highly sensitive optical setups to overcome the limitations of the readout. Here, we demonstrate a flexible strategy for assembling highly luminescent and colorimetric quantum dot-nucleic acid hairpin (QD-HP) molecular beacons for use in CRISPR/Cas diagnostics. This strategy utilizes a chimeric peptide-peptide nucleic acid (peptide-PNA) to conjugate fluorescently labeled DNA or RNA hairpins to ZnS-coated QDs. QDs are particularly promising alternatives for molecular beacons due to their greater brightness, strong UV absorbance with large emission offset, exceptional photostability, and potential for multiplexing due to their sharp emission peaks. Using Förster resonance energy transfer (FRET), we have developed ratiometric reporters capable of pM target detection (without nucleotide amplification) for both target DNA and RNA, and we further demonstrated their capabilities for multiplexing and camera-phone detection. The flexibility of this system is imparted by the dual functionality of the QD as both a FRET donor and a central nanoscaffold for arranging nucleic acids and fluorescent acceptors on its surface. This method also provides a generalized approach that could be applied for use in other CRISPR/Cas nuclease systems.
利用CRISPR/Cas核酸酶Cas13和Cas12的策略在开发用于检测致病核酸的高灵敏度快速诊断检测方法方面显示出巨大潜力。最常见的利用荧光团-淬灭剂分子信标的方法需要链扩增策略或高灵敏度光学装置来克服检测的局限性。在此，我们展示了一种灵活的策略，用于组装高发光和比色量子点-核酸发夹（QD-HP）分子信标，用于CRISPR/Cas诊断。该策略利用嵌合肽-肽核酸（肽-PNA）将荧光标记的DNA或RNA发夹与硫化锌包覆的量子点偶联。量子点因其更高的亮度、具有大发射偏移的强紫外吸收、出色的光稳定性以及由于其尖锐发射峰而具有的多路复用潜力，是分子信标的特别有前景的替代品。利用福斯特共振能量转移（FRET），我们开发了能够对目标DNA和RNA进行pM级目标检测（无需核苷酸扩增）的比率报告分子，并进一步展示了它们的多路复用和手机摄像头检测能力。该系统的灵活性源于量子点的双重功能，即作为FRET供体和用于在其表面排列核酸和荧光受体的中央纳米支架。该方法还提供了一种可应用于其他CRISPR/Cas核酸酶系统的通用方法。
