A nonfluorescent, broad-range quencher dye for Forster resonance energy transfer assays
A nonfluorescent, broad-range quencher dye for Forster resonance energy transfer assays
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DOI：
10.1016/j.ab.2009.02.024
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发表时间：
2009-05-15
影响因子：
2.9
通讯作者：
Olive, D. Michael
中科院分区：
文献类型：
--
作者：
Peng, Xinzhan;Chen, Huaxian;Olive, D. Michael
我们在这里报告一种新的，水溶性的，非荧光染料，有效地淬灭荧光从广泛的可见光和近红外（NIR）荧光团在福斯特共振能量转移（FRET）系统。使用基于FRET的半胱天冬酶-3测定系统模型来测试猝灭剂染料的性能。通过将猝灭剂IRDye（（R））QC-1与GDEVDGAK肽结合荧光素制备荧光半胱天冬酶-3底物（发射最大值类似于540 nm），Cy 3在一些实施方案中，所述光致发光材料包括Cy 5（类似于570 nm）、Cy 5（类似于670 nm）、IRDye 680（类似于700 nm）、IRDye 700DX（类似于690 nm）或IRDye 800 CW（类似于790 nm）。对于这些染料/猝灭剂对，福斯特距离R-0值计算为41至65 A。通过测定重组caspase-3完全裂解后的荧光变化来测定这些肽的荧光猝灭效率，其范围为97.5%~ 98.8%。荧光底物的半胱天冬酶裂解的荧光倍数增加范围为40至83，这取决于染料/猝灭剂对。因为IRDye QC-1有效地猝灭了NIR荧光团（例如，IRDye 700DX、IRDye 680、IRDye 800 CW）和可见荧光团（例如，荧光素，Cy 3，Cy 5），其在使用多种荧光染料的FRET测定中具有广泛的适用性。(c)2009 Elsevier Inc. All rights reserved.
We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Forster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 Substrates were prepared by conjugating the quencher, IRDye((R)) QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum similar to 540 nm), Cy3 (similar to 570 nm), Cy5 (similar to 670 nm), IRDye 680 (similar to 700 nm), IRDye 700DX (similar to 690 mn), or IRDye 800CW (similar to 790 nm). The Forster distance R-0 values are calculated as 41 to 65 A for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change oil complete cleavage by recombinant caspase-3 and ranged front 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes. (c) 2009 Elsevier Inc. All rights reserved.