Canzano Mary, Harman Joseph L, Méndez Yanira, Quijano-Rubio Alfredo, Silva Daniel-Adriano, Bernardes Gonçalo J L
Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.
Monod Bio Inc., Seattle, Washington 98109, United States.
JACS Au. 2025 Jul 28;5(8):3914-3925. doi: 10.1021/jacsau.5c00576. eCollection 2025 Aug 25.
Rapid, accurate, and accessible diagnostics for pathogenic infections are of vital importance for the prevention of disease transmission and mitigation of future pandemics. Biosensors employing the CRISPR nuclease Cas13 have enabled robust detection of viral RNA. However, existing Cas13-based diagnostics primarily utilize fluorescent or lateral flow assay (LFA) readouts, impeding detection in complex sample media. Here, we have developed a luciferase-based Cas13 diagnostic containing a protein-nucleic acid conjugate, coined Selective One-pot LUminescent Nucleic Acid Reporter (SOLUNAR). SOLUNAR utilizes RNA which has been site-selectively bioconjugated to block the binding of a de novo heterodimeric protein pair. In the presence of a viral target, the sensor RNA is cleaved by the collateral activity of Cas13, restoring binding and yielding a luminescent signal by the reconstitution of a tagged de novo split luciferase. An integral component of SOLUNAR, the use of de novo proteins enabled precise engineering of site-selective chemical handles and tunable binding affinities to modulate activity. SOLUNAR selectively detects Influenza A H1N1 at room temperature in one-pot with a limit of detection (LOD) of 1.5 nM without amplification and is compatible with samples containing ≤10% serum. Our findings demonstrate the utility of luminescence in Cas13 diagnostics and reveal the potential applications of this platform to activity-based enzymatic sensing.
针对致病性感染的快速、准确且易于获取的诊断方法对于预防疾病传播和减轻未来大流行至关重要。采用CRISPR核酸酶Cas13的生物传感器能够对病毒RNA进行可靠检测。然而，现有的基于Cas13的诊断方法主要利用荧光或侧向流动分析（LFA）读数，这在复杂样本介质中阻碍了检测。在此，我们开发了一种基于荧光素酶的Cas13诊断方法，其中包含一种蛋白质 - 核酸共轭物，称为选择性一锅发光核酸报告器（SOLUNAR）。SOLUNAR利用经过位点选择性生物共轭的RNA来阻断新生异二聚体蛋白对的结合。在存在病毒靶标的情况下，传感器RNA被Cas13的旁系活性切割，恢复结合并通过重组标记的新生分裂荧光素酶产生发光信号。SOLUNAR的一个重要组成部分是，使用新生蛋白能够对位点选择性化学手柄进行精确工程设计，并具有可调的结合亲和力以调节活性。SOLUNAR在室温下一锅法中选择性地检测甲型H1N1流感，检测限（LOD）为1.5 nM，无需扩增，并且与含≤10%血清的样本兼容。我们的研究结果证明了发光在Cas13诊断中的实用性，并揭示了该平台在基于活性的酶传感中的潜在应用。
