Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA.
Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA.
Sci Signal. 2022 Aug 16;15(747):eabq7618. doi: 10.1126/scisignal.abq7618.
The intrinsic fluorescence of samples confounds the use of fluorescence-based sensors. This is of particular concern in high-throughput screening (HTS) applications using large chemical libraries containing intrinsically fluorescent compounds. To overcome this problem, we developed a bioluminescence resonance energy transfer (BRET) Ca sensor, CalfluxCTN. We demonstrated that it reliably reported changes in intracellular Ca concentrations evoked by an agonist and an antagonist of the human muscarinic acetylcholine receptor M (hMR) even in the presence of the fluorescent compound fluorescein, which interfered with a standard fluorescent HTS sensor (Fluo-8). In an HTS using a chemical library containing fluorescent compounds, CalfluxCTN accurately identified agonists and antagonists that were missed or miscategorized using Fluo-8. Moreover, we showed that a luciferase substrate that becomes activated only when inside cells generated long-lasting BRET signals in HTS, enabling results to be reliably compared among replicate samples for hours. Thus, the use of a self-luminescent sensor instead of a fluorescent sensor could facilitate the complete screening of chemical libraries in a high-throughput context and enable analysis of autofluorescent samples in many different applications.
样品的固有荧光会干扰基于荧光的传感器的使用。在使用包含固有荧光化合物的大型化学文库进行高通量筛选 (HTS) 应用时，这尤其令人关注。为了克服这个问题，我们开发了一种生物发光共振能量转移 (BRET) Ca 传感器，CalfluxCTN。我们证明，即使在存在荧光化合物荧光素的情况下，它也能可靠地报告人毒蕈碱乙酰胆碱受体 M (hMR) 的激动剂和拮抗剂引起的细胞内 Ca 浓度变化，而荧光素会干扰标准荧光 HTS 传感器 (Fluo-8)。在使用包含荧光化合物的化学文库进行的 HTS 中，CalfluxCTN 准确地识别了使用 Fluo-8 错过或分类错误的激动剂和拮抗剂。此外，我们还表明，只有当细胞内激活时才会被激活的荧光素酶底物会在 HTS 中产生持久的 BRET 信号，从而能够可靠地比较数小时内重复样本的结果。因此，使用自发光传感器而不是荧光传感器可以促进在高通量环境下对化学文库的全面筛选，并能够在许多不同的应用中分析自发荧光样品。
