New tool lets scientists observe genome dynamics in real time

A technique to see DNA as it moves in living cells could offer novel fundamental insights into biology.

The human genome is like a big ball of yarn, made up of 3 billion molecular units arranged in sequence and then wrapped up around itself. Within this ball of yarn are your genes, which are regions of DNA that get copied and then turned into miniature molecular machines called proteins. The three-dimensional structure of the yarn dictates which of your genes get turned into proteins, and when this system fails, disease develops. But until now, it has not been possible to visualize how different regions of DNA talk to each other across space and time. 

A team at Stanford led by Stanley Qi, associate professor of bioengineering in the Schools of Engineering and Medicine and Institute Scholar at Sarafan ChEM-H, and W.E. Moerner, Harry S. Mosher professor of chemistry in the School of Humanities and Sciences, combined their expertise in DNA technology and super-resolution imaging to develop a new tool that can light up any region of the genome in any living cell and enable scientists to watch how different regions interact with one another. The tool, reported in Cell on April 15, could help scientists understand how certain genes get turned on and off in healthy cells and in diseases like cancer.

Previously, researchers were only able to see snapshots of DNA interactions at different timepoints in preserved cells. Much like the difference between a photograph and a video, their technology reveals a fourth dimension – time – in cells that are alive and dynamically changing. 

“Our work turns Instagram into YouTube,” said Qi. “It gives a direct understanding of what’s going on over time in cells.” 

Of particular interest are regions of DNA that do not contain genes. Only about 2% of the 3 billion units in our DNA ever get converted into proteins, a process known as gene expression. Scientists used to refer to the other 98% as junk DNA, since it did not seem to have a clear purpose. We now know that these mysterious DNA regions contain important features that control gene expression. 

“They are like the software controlling the DNA program,” said Qi. 

These detailed movies of previously overlooked pieces of genome could offer new fundamental insights into biology. In addition, seeing how the “software” changes in real time in healthy cells compared to diseased ones could provide clues about faulty gene expression related to illness. 

Fluorescent mailmen

The first step in making this tool was figuring out how to observe a specific region of the genome within the abundant complexity of the DNA yarn ball. For this, the researchers turned to a version of the gene editing technology called CRISPR. This version of CRISPR uses an engineered protein called dCas9, together with an RNA molecule that acts as a mailing address for a particular site in the genome. This enables the dCas9-RNA complex to act like a mailman, finding and attaching itself to one desired DNA address – all while carrying a fluorescent dye molecule that is visible as light under the microscope. 

The researchers made their fluorescent signal brighter by sending a flock of molecular mailmen to span unique DNA addresses within the same genetic zip code, lighting up the DNA “street” of any gene they wanted to study. Under a traditional light microscope, this activity would look like a blurry blob because the movements of DNA are on the order of tens of nanometers – 5,000 times smaller than the width of a human hair and well below what a traditional light microscope can resolve. To overcome this limitation, the Qi lab turned to the expertise of Moerner and his team, who helped pioneer techniques to detect light emitted from single fluorescent molecules, a feat that earned Moerner the Nobel Prize in Chemistry in 2014.

Super-resolution microscopy techniques to detect light from single molecules are now widely used, but a limitation of many microscopes is the inability to collect information about how a molecule moves in all three dimensions at the same time. Scientists can watch a piece of DNA jiggle left and right, but the up and down motion would only be captured in snapshots that miss big chunks of time. To fill in the gaps, Moerner and team use an optical trick in their microscope to extract simultaneous position information about DNA in all three dimensions at once. Much like a prism can split visible light into its components to produce a rainbow, passing light through other materials causes it to shapeshift in different ways. 

“There are actually a lot of amazing things that can be done with light. What we did was add a special optical component that re-scrambles one spot of light into two spots, so that depth information is encoded in the angle between the two spots,” Moerner explained. 

Of course, those spots are the fluorescent mailmen that the team appended to DNA. And the angle is the critical missing piece of spatial information that allowed the researchers to capture the full picture of DNA architecture in real time.

Visualizing DNA movement during transcription 

The team put their molecular mailmen to the test by having them follow the “software” – formerly part of the “junk” DNA – that controls the process of copying genes before they get turned into proteins, called transcription. They found that these regulatory DNA regions nestled closer together and jiggled less during transcription, as if talking to each other. Just what language the DNA is speaking, what other molecules might be involved, and whether this happens with all genes or just a few, are topics ripe for future study. 

“Transcription itself is fundamental to biology, and we’ve got this nanoscale insight in a way that not many get the chance to see,” said study author Ashwin Balaji, a graduate student in the Moerner lab. 

Because their tool can be delivered into any living cell, they can use it to image the genomes of primary cells – cells isolated directly from tissues that help scientists understand what happens in the body. 

“When we can see different sites of DNA in primary cells, like neurons and immune cells, that makes me very excited because it hasn’t been seen before,” said first author Yanyu Zhu, postdoctoral scholar in the Qi lab. 

In the future, the technology could also be used to study cells directly from patients, such as from tumor biopsies, and could be particularly valuable for understanding how regulatory regions of DNA that do not code for genes contribute to disease.

“We are trying to learn the secret behind the 98% ‘junk’ DNA,” said Qi. “No one calls it junk anymore because we know it is very important, but we still lack so much information about what it does, and most importantly, how it plays a role in disease.” 

The Stanford team has worked to make their technology accessible to other researchers by making their design and analysis algorithms freely available. The development of these tools was also assisted by a third lab at Stanford led by Andrew Spakowitz, professor of chemical engineering and materials science, making this a truly interdisciplinary effort. 

“I find these kinds of collaborations to be very powerful because you can go further than either group alone,” said Moerner. “People bring different skills to the table, which makes it very stimulating, very exciting.”

Qi is also a member of Bio-X, the Cardiovascular Institute, the Maternal & Child Health Research Institute (MCHRI), the Stanford Cancer Institute, the Wu Tsai Neurosciences Institute, and is a Chan Zuckerberg Biohub – San Francisco Investigator. Moerner is also a professor, by courtesy, of applied physics, a faculty fellow of Sarafan ChEM-H, and a member of Bio-X and of the Wu Tsai Neurosciences Institute. Spakowitz is also a professor, by courtesy, of applied physics and chemistry and a member of Bio-X and the Institute for Computational and Mathematical Engineering. Other Stanford co-authors include postdoctoral scholar Yanyu Zhu, PhD student Ashwin Balaji, postdoctoral scholar Mengting Han, postdoctoral scholar Leonid Andronov, former PhD student Anish Roy, PhD student Crystal Chen, postdoctoral scholar Leanne Miles, PhD student Sa Cai, undergraduate student Zhengxi Gu, PhD student Ariana Tse, and postdoctoral scholar Takeshi Uenaka. 

This work was supported by the National Institutes of Health, the National Science Foundation, the Stanford School of Medicine, Bio-X, and the V Foundation.

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Media contact: Rebecca McClellan, Sarafan ChEM-H: rmcclell [at] stanford.edu (rmcclell[at]stanford[dot]edu)