The human genome can be likened to an intricate ball of yarn, intricately woven from approximately 3 billion molecular units meticulously arranged in a sequence that often coils around itself. Within this complex structure lie genessegments of DNA that are pivotal to the formation of proteins, the miniature molecular machines that facilitate vital biological functions. The three-dimensional arrangement of this genetic yarn determines which genes remain activated and subsequently transformed into proteins. When this elaborate system experiences malfunctions, various diseases can emerge. For many years, scientists have grappled with the challenge of visualizing the dynamic communication between different regions of DNA over time.

In a remarkable breakthrough, a team of researchers at Stanford University, led by Stanley Qi, an associate professor of bioengineering, in collaboration with W.E. Moerner, a distinguished professor of chemistry, has developed an innovative tool. This novel imaging technology enables scientists to illuminate any selected region of the genome within living cells, allowing for real-time observation of interactions between different DNA regions. The groundbreaking findings of this study were published in the prestigious journal Cell on April 15, and they have the potential to significantly enhance our understanding of gene regulation in both healthy tissues and diseases, including cancer.

Traditionally, researchers have been restricted to capturing static snapshots of DNA interactions at various moments, akin to comparing photographs with videos. The new technology introduced by Qi and his colleagues adds a fourth dimensiontimebringing to life the dynamic changes occurring within living cells.
Our work turns Instagram into YouTube, Qi remarked, emphasizing how this advancement offers a direct and comprehensive understanding of cellular processes over time.

A major focus of this research is the non-coding regions of DNA, often historically dismissed as junk DNA. In reality, only about 2% of the 3 billion DNA units are utilized to code for proteins through a process known as gene expression. The remaining 98% was frequently regarded as functionless. However, contemporary research has unveiled that these seemingly redundant segments of DNA harbor crucial elements that play significant roles in regulating gene expression.
They function like the software that controls the DNA program, Qi explained, underscoring the importance of these non-coding regions.

The capability to produce detailed visualizations of these neglected areas of the genome could yield groundbreaking insights in biology. Furthermore, studying how this regulatory software adapts in healthy versus diseased cells may illuminate the underlying mechanisms of abnormal gene expression associated with various illnesses.

To develop their imaging tool, the researchers first formulated a strategy to examine specific areas within the overwhelmingly complex DNA structure. They employed a refined version of CRISPR gene-editing technology, using an engineered protein known as dCas9, accompanied by an RNA molecule that guides the protein to target sites within the genome. The dCas9-RNA complex operates much like a mailman, locating and attaching to a specific DNA address while carrying a fluorescent dye molecule that emits visible light under a microscope.

To enhance the brightness of their fluorescent signals, the team deployed multiple molecular mailmen to illuminate unique DNA addresses within the same genetic region, effectively lighting up any gene they aimed to investigate. When viewed under conventional light microscopy, this activity may appear as a blurry mass due to the nanometer-scale movements of DNA, which are 5,000 times smaller than a human hair, beyond the resolution capabilities of standard light microscopes. To overcome these limitations, the Qi lab partnered with Moerner's team, renowned for pioneering methods to detect light emitted from individual fluorescent moleculesa breakthrough that earned Moerner the Nobel Prize in Chemistry in 2014.

Although super-resolution microscopy techniques have gained popularity, many microscopes struggle to capture simultaneous information about a molecule's movement in all three spatial dimensions. While scientists can observe lateral movements of DNA, the up-and-down motions usually get lost to time, recorded only in fragmented snapshots. To rectify this, Moerner and his team introduced an optical innovation to their microscope that enables the simultaneous extraction of positional data regarding DNA in three dimensions. Much like how a prism can disperse light to produce a spectrum, these researchers manipulated light to encode depth information based on angles formed between two light sources.

There are indeed many remarkable applications of light, Moerner elaborated. We introduced a specialized optical element that reorganizes a single point of light into two, thereby encoding depth information in the angle between the two.

The researchers subsequently tested their molecular mailmen by tracking the regulatory software within previously considered junk DNA that governs the transcription processwhere genes are copied before being transformed into proteins. Their findings revealed that these regulatory regions positioned themselves closer together and exhibited less movement during transcription, suggesting potential communication between them. The nature of this dialogue among DNA regions, the involvement of other molecules, and whether this pattern holds across all genes are intriguing questions that could guide future research.

Transcription is a fundamental biological process, and this nanoscale insight is something few have had the opportunity to observe, remarked Ashwin Balaji, a graduate student in Moerner's lab and co-author of the study.

The versatility of their tool allows it to be delivered into any living cell, enabling imaging of the genomes of primary cellsthose isolated directly from tissueswhich is crucial for grasping biological processes within the body.
The ability to visualize different DNA sites within primary cells, such as neurons and immune cells, excites me tremendously, as this is unprecedented, expressed Yanyu Zhu, the first author of the study and a postdoctoral scholar in Qi's lab.

Looking forward, this innovative technology could be utilized to investigate cells extracted from patients, such as tumor biopsies, providing invaluable insights into how the regulatory regions of DNA that do not code for genes contribute to various diseases.
We are striving to uncover the secrets behind the 98% of DNA once deemed junk, Qi stated. No one refers to it as junk anymore, given its recognized importance, yet we still have so much to learn about its functions and roles in diseases.

To promote accessibility for other researchers, the Stanford team has made their design and analysis algorithms publicly available. Their collaborative efforts were further enhanced by contributions from a third Stanford laboratory led by Andrew Spakowitz, a professor of chemical engineering and materials science, highlighting a truly interdisciplinary approach to this important research.
I find these collaborations immensely powerful because they enable us to achieve far more than any individual group could alone, Moerner concluded. The diverse skill sets that each team brings create a stimulating and exciting environment for scientific exploration.