Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapogu, Seoul 04107, Korea.
Chemistry Education Program, Department of Mathematics and Science Education, Sanata Dharma University, Yogyakarta 55282, Indonesia.
Molecules. 2022 Aug 17;27(16):5248. doi: 10.3390/molecules27165248.
Fluorescent protein-DNA-binding peptides or proteins (FP-DBP) are a powerful means to stain and visualize large DNA molecules on a fluorescence microscope. Here, we constructed 21 kinds of FP-DBPs using various colors of fluorescent proteins and two DNA-binding motifs. From the database of fluorescent proteins (FPbase.org), we chose bright FPs, such as RRvT, tdTomato, mNeonGreen, mClover3, YPet, and mScarlet, which are four to eight times brighter than original wild-type GFP. Additionally, we chose other FPs, such as mOrange2, Emerald, mTurquoise2, mStrawberry, and mCherry, for variations in emitting wavelengths. For DNA-binding motifs, we used HMG (high mobility group) as an 11-mer peptide or a 36 kDa tTALE (truncated transcription activator-like effector). Using 21 FP-DBPs, we attempted to stain DNA molecules and then analyzed fluorescence intensities. Most FP-DBPs successfully visualized DNA molecules. Even with the same DNA-binding motif, the order of FP and DBP affected DNA staining in terms of brightness and DNA stretching. The DNA staining pattern by FP-DBPs was also affected by the FP types. The data from 21 FP-DBPs provided a guideline to develop novel DNA-binding fluorescent proteins.
荧光蛋白-DNA 结合肽或蛋白（FP-DBP）是在荧光显微镜上染色和可视化大 DNA 分子的有力手段。在这里，我们使用各种颜色的荧光蛋白和两个 DNA 结合基序构建了 21 种 FP-DBP。从荧光蛋白数据库（FPbase.org）中，我们选择了 RRvT、tdTomato、mNeonGreen、mClover3、YPet 和 mScarlet 等明亮的 FP，其亮度比原始野生型 GFP 亮四到八倍。此外，我们选择了 mOrange2、Emerald、mTurquoise2、mStrawberry 和 mCherry 等其他 FP，以改变发射波长。对于 DNA 结合基序，我们使用 HMG（高迁移率族）作为 11 肽或 36 kDa tTALE（截断转录激活样效应物）。使用 21 种 FP-DBP，我们尝试染色 DNA 分子，然后分析荧光强度。大多数 FP-DBP 成功地可视化了 DNA 分子。即使使用相同的 DNA 结合基序，FP 和 DBP 的顺序也会影响 DNA 染色的亮度和 DNA 拉伸程度。FP-DBP 的 DNA 染色模式也受到 FP 类型的影响。来自 21 种 FP-DBP 的数据为开发新型 DNA 结合荧光蛋白提供了指导。
