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.

在核基因组中对短片段非重复DNA进行原位高分辨可视化，对于研究单细胞内的环化相互作用与染色质组织至关重要。近年来，基于Oligopaints探针的荧光原位杂交（FISH）技术取得进展，可实现分辨率极限为4.9 kb的基因组区域超分辨率成像。为靶向更短的基因组元件，我们开发了一种仅使用分子信标（MB）探针的简易FISH方法，该方法可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（3D-STORM）并优化成像条件，实现了稀疏分布的Alexa-647与细胞自身背景自发荧光的高效区分。借助3D-STORM技术与29至34条独立MB探针，我们分别观测到人类及小鼠基因组中2.5 kb的整合型或内源性独特DNA的原位3D精细纳米结构，证明了基于MB探针的FISH技术可在超分辨率水平下实现迄今最短的非重复基因组序列的三维可视化。