University of Idaho, Dept of Chemistry, 875 Perimeter Dr, Moscow, ID 83844-2343, USA.
Biotechniques. 2019 Mar;66(3):121-127. doi: 10.2144/btn-2018-0128. Epub 2019 Feb 15.
To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.
为了详细探讨热荧光分析（TFA），我们比较了两种相关的适体。第一个是 LINN2，它是一种针对 EGFR 重组蛋白的 DNA 适体。在这项工作中，我们选择了第二个适体 KM4，它针对高表达 EGFR 的 A549 细胞。这两种适体来自同一个文库，与相同的靶标结合，但在 TFA 中表现不同。我们的结果提出了关于适体 TFA 的四个总体结论：1. 一些适体在与靶标结合时显示荧光降低，表明靶标结合的适体并不总是荧光的。2. 许多适体并不遵循适体-靶标相互作用稳定折叠构象的直观假设。3. TFA 可能最适合具有显著双链结构的适体。4. 动力学效应可能很重要，并且在准备样品时的操作顺序应仔细优化。
