By studying how individual DNA molecules behave, we can better understand genetic disorders and design better medicines. Until now, however, examining DNA molecules one by one has been a slow process. Biophysicists from Delft University of Technology and Leiden University developed a technique that speeds up the screening of individual DNA molecules at least a thousand times. With this technology they can measure millions of DNA molecules within a week instead of years to decades. The research has now been published in Science.
“DNA, RNA and proteins are the most important players in regulating all processes in the cells of our body,” explains Leiden professor John van Noort. “To understand the (mis)function of these molecules, it is essential to discover how their 3D structure depends on their sequence and for this it is necessary to measure them molecule by molecule. However, measurements of single molecules are laborious and slow, and the number of possible sequence variations is enormous.”
From decades to days
Now the team of scientists has developed an innovative tool called SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space), which can study millions of DNA molecules simultaneously. “Traditional techniques that allow one sequence to be examined at a time usually require hours of measuring 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, providing new insights into how the structure and function of DNA depend on the sequence. Furthermore, the technique can be used to quickly find the best sequence for applications ranging from nanotechnology to personalized medicine “, says PhD candidate Carolien Bastiaanssen.
Never combined before
To create their new SPARXS technique, the researchers combined two existing technologies that had never been combined before: single-molecule fluorescence and next-generation Illumina sequencing. In the first technique, molecules are labeled with a fluorescent dye and made visible with a sensitive microscope. This latter technique reads millions of DNA codes simultaneously. Joo: “It took a year to determine whether combining the two techniques is feasible, another four years to develop a method, and another two years to ensure the accuracy and consistency of the measurements while at the same time manage the enormous amount of data generated.”
“The really fun and interesting part started when we had to interpret the data,” says first author Ivo Severins. “Since these experiments, which combine single-molecule measurements with sequencing, are completely new, we had no idea what results we would and could obtain. It took 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 dealing with the large amount of data, Van Noort adds: ‘We had to develop an automated and robust analysis pipeline. This was especially challenging because individual molecules are fragile and yield only a small amount of light, so the resulting data do not provide direct insight into how sequence affects the structure and dynamics of DNA, even for the relatively simple DNA structures that We have studied our knowledge of DNA structure, and compared it with the experimental data.”
Medical advances
More precise manipulation and understanding of DNA sequences will likely lead to advances in medical treatments, such as more effective gene therapies and personalized medicine. The researchers also envision biotechnological innovations and a better understanding of biology at the molecular level. Joo: “We expect that applications in genetic research, drug development and biotechnology will emerge within the next five to ten years.”
