Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes
- College of Optoelectronic Engineering, Shenzhen University, China
- Tsinghua University, China
- The University of Hong Kong, China
Abstract
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.
Data availability
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Data from: Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probesAvailable at Dryad Digital Repository under a CC0 Public Domain Dedication.
Article and author information
Author details
Michael Qiwei Zhang
Funding
National Natural Science Foundation of China (61235012)
- Hanben Niu
National Natural Science Foundation of China (61335001)
- Michael Qiwei Zhang
Shenzhen Science and Technology Planning Project (JCYJ20150324141711698)
- Michael Qiwei Zhang
National Natural Science Foundation of China (31401146)
- Yanxiang Ni
National Natural Science Foundation of China (31361163004)
- Michael Qiwei Zhang
National Natural Science Foundation of China (91019016)
- Michael Qiwei Zhang
the National Basic Research Program of China (2012CB825802)
- Hanben Niu
the Special-Funded Program on National Key Scientific Instruments and Equipment Development (2012YQ150092)
- Hanben Niu
the National Key Basic Research Project (2012CB316503)
- Michael Qiwei Zhang
the National Key Basic Research Program of China (2015CB352005)
- Hanben Niu
National Natural Science Foundation of China (61178080)
- Michael Qiwei Zhang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Ni et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes
eLife 6:e21660.
https://doi.org/10.7554/eLife.21660
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