Data from: Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaints probes enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses only molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) and optimized the imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and 29-34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in the human or mouse genome, respectively, demonstrating the capability of MB-based FISH in visualizing a so far shortest and non-repetitive genomic sequence in 3D at super-resolution.

在核基因组中对原位短非重复DNA开展高分辨可视化研究，对于解析单细胞内的染色质环相互作用与染色质组织具有至关重要的意义。此前基于Oligopaints探针的荧光原位杂交（fluorescence in situ hybridization, FISH）技术已实现分辨率极限达4.9千碱基对（kb）的基因组结构域超分辨成像。为实现更短基因组元件的靶向成像，我们开发了一种简易的荧光原位杂交方法：仅使用分子信标（molecular beacon, MB）探针促进探针与靶标结合，同时最大限度降低非特异性荧光信号。我们采用三维随机光学重构显微镜（three-dimensional stochastic optical reconstruction microscopy, 3D-STORM）并优化成像条件，可有效区分稀疏分布的Alexa-647荧光信号与细胞自发荧光背景。结合三维随机光学重构显微镜与29至34条独立分子信标探针，我们分别在人类与小鼠基因组中，原位观测到了2.5千碱基对的整合型或内源性单拷贝独特DNA的三维精细纳米结构。上述结果证实，基于分子信标的荧光原位杂交技术能够以超分辨水平实现3D可视化迄今最短的非重复基因组序列。