Yuan Aijiao, Sun Tianrui, Chen Leyuan, Zhang Dapeng, Xie Wenjing, Peng Hanyong
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2024 Dec 17;96(50):20074-20081. doi: 10.1021/acs.analchem.4c04993. Epub 2024 Dec 5.
Circulating tumor nucleic acids (CTNAs), which consist of cell-free DNA or RNA released from tumor cells, are utilized as potential biomarkers for diagnosing and managing tumor prognosis. There is a significant demand for developing a highly sensitive and reliable assay for CTNAs detection. In this study, we engineered a CRISPR/Cas12a corona nanomachine capable of detecting circulating tumor DNA and RNA in serum. This nanomachine consists of a protein shell incorporating Cas12a/crRNA ribonucleoprotein complexes and a scaffold AuNP core decorated with substrate ssDNA strands. The protective CRISPR corona shields the nucleic acid core from degradation by nuclease DNase/RNase, thereby enhancing the stability of the CRISPR/Cas12a corona nanomachine in biological fluids, even tolerating up to undiluted human serum and FBS. Upon encountering target CTNAs, the CRISPR/Cas12a is activated through the sequence-specific hybridization between crRNA and CTNAs. Subsequently, the activated CRISPR/Cas12a autonomously cleaves the collateral ssDNA substrates on AuNPs, releasing the fluorophore-labeled fragment and generating an increasing fluorescent signal. The CRISPR corona nanomachine was successfully employed to detect various CTNAs, including circulating tumor (ct)DNA/RNA ( L858R) and microRNA-21, achieving a limit of detection of 0.14 pM for ctDNA and 1.0 pM for RNA. This CRISPR corona nanomachine enables simultaneous detection of both DNA and RNA in complex biological samples, offering a promising tool for early diagnosis.
循环肿瘤核酸（CTNAs）由肿瘤细胞释放的游离DNA或RNA组成，被用作诊断和管理肿瘤预后的潜在生物标志物。开发一种用于CTNAs检测的高度灵敏且可靠的检测方法有很大需求。在本研究中，我们构建了一种能够检测血清中循环肿瘤DNA和RNA的CRISPR/Cas12a冠状纳米机器。这种纳米机器由一个包含Cas12a/crRNA核糖核蛋白复合物的蛋白质外壳和一个装饰有底物单链DNA的支架金纳米颗粒核心组成。保护性的CRISPR冠层可保护核酸核心不被核酸酶DNase/RNase降解，从而增强CRISPR/Cas12a冠状纳米机器在生物流体中的稳定性，甚至能耐受高达未稀释的人血清和胎牛血清。遇到目标CTNAs时，CRISPR/Cas12a通过crRNA与CTNAs之间的序列特异性杂交被激活。随后，激活的CRISPR/Cas12a自主切割金纳米颗粒上的旁系单链DNA底物，释放出荧光团标记的片段并产生不断增加的荧光信号。CRISPR冠状纳米机器成功用于检测各种CTNAs，包括循环肿瘤（ct）DNA/RNA（L858R）和微小RNA-21，实现了对ctDNA的检测限为0.14 pM，对RNA的检测限为1.0 pM。这种CRISPR冠状纳米机器能够同时检测复杂生物样品中的DNA和RNA，为早期诊断提供了一种有前景的工具。
