Studying how single DNA molecules behave helps us to better understand genetic disorders and design better drugs. Until now however, examining DNA molecules one-by-one was a slow process. Biophysicists from Delft University of Technology and Leiden University developed a technique that speeds up 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. DNA, RNA and proteins are the key players to regulate all processes in the cells of our body. To understand the (mis-)functioning of these molecules, it is essential to uncover how their 3D structure depends on their sequence and for this it is necessary to measure them one molecule at a time. However, single-molecule measurements are laborious and slow, and the number of possible sequence variations is massive.

 

Now the team of scientists developed an innovative tool, called SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space), that allows for studying millions of DNA molecules simultaneously. "Traditional techniques that allow one sequence to be probed at a time usually take 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 enables us to study large sequence libraries, providing new insights into how the structure and function of DNA depend on sequence. Additionally, the technique can be used to quickly find the best sequence for applications ranging from nanotechnology to personalised medicine. To create their new SPARXS technique, the researchers combined two existing technologies that had never been paired before: single-molecule fluorescence and next-generation Illumina sequencing. In the first technique, molecules are labelled with a fluorescent dye and visualised using a sensitive microscope. The latter technique reads out millions of DNA codes simultaneously.

 

Another challenge they had to overcome was handling the large amount of data, Van Noort adds: "We had to develop an automated and robust analysis pipeline. This was particularly challenging as single molecules are fragile and yield only a tiny amount of light, making the data inherently noisy. Furthermore, the resulting data do not directly provide insights into how the sequence affects the structure and dynamics of DNA, even for the relatively simple DNA structures that we studied. To really test our understanding, we set up a model that incorporates our knowledge of the DNA structure, and compared it with the experimental data."