From decades to days: Biophysicists reveal DNA behavior in record time

Studying the behavior of individual DNA molecules helps us better understand genetic disorders and develop better drugs. Until now, however, studying individual DNA molecules has been a lengthy process.

Biophysicists from Delft University of Technology and Leiden University have developed a technique that speeds up the study of individual DNA molecules by at least a thousand times. This technology allows them to measure millions of DNA molecules within a week, rather than after years or decades. The study was published in Science.

“DNA, RNA and proteins are the main players in regulating all processes in the cells of our body,” explains Leiden professor John van Noort.

“To understand how these molecules (mal)function, one must find out how their 3D structure depends on their sequence. To do this, it is necessary to measure them molecule by molecule. However, single-molecule measurements are laborious and slow, and the number of possible sequence variations is enormous.”

Now the team of scientists has developed an innovative tool called SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space), which enables the simultaneous investigation of millions of DNA molecules.

“Conventional techniques that can only examine one sequence at a time usually require several hours of measurement time per sequence. With SPARXS, we can measure millions of molecules within a day to a week. Without SPARXS, such a measurement would take several years to decades,” says Delft professor Chirlmin Joo.

“SPARXS allows us to study large sequence libraries and provides new insights into how the structure and function of DNA depends on the sequence. In addition, the technique can be used to quickly find the best sequence for applications ranging from nanotechnology to personalized medicine,” adds PhD student Carolien Bastiaanssen.

Never combined

To develop their new SPARXS technique, the researchers combined two existing technologies that had never been combined before: single-molecule fluorescence and Illumina next-generation sequencing.

The first technique involves marking molecules with a fluorescent dye and making them visible using a sensitive microscope. The second technique allows millions of DNA codes to be read simultaneously.

Joo says: “It took a year to determine whether combining the two techniques was feasible, another four years to develop a working method, and another two years to ensure accuracy and consistency of measurements while managing the enormous amount of data generated.”

“The really fun and interesting part started when we had to interpret the data,” says lead author Ivo Severins.

“Because these experiments, which combine single-molecule measurements with sequencing, are completely new, we had no idea what results we would and could get. It required a lot of searching through the data to find correlations and patterns and to determine the mechanisms underlying the patterns we see.”

Overcoming data processing challenges

Another challenge they had to overcome was processing the large amount of data, Van Noort adds: “We had to develop an automated and robust analysis pipeline. This was particularly challenging because individual molecules are fragile and emit only a small amount of light, which makes the data inherently noisy.”

“In addition, the data we obtained do not provide direct insights into the effect of sequence on the structure and dynamics of DNA, even for the relatively simple DNA structures we studied. To really test our understanding, we built a model that incorporates our knowledge of DNA structure and compared it with the experimental data.”

More precise manipulation and understanding of DNA sequences will likely lead to advances in medical treatment, such as more effective gene therapies and personalized medicine. Researchers also expect biotechnology innovations and an overall better understanding of biology at the molecular level.

Joo concludes: “We expect that within the next five to ten years we will see initial applications in genetic research, drug development and biotechnology.”