Studying the behavior of individual DNA molecules is crucial for enhancing our understanding of genetic disorders and developing improved drugs. Historically, analyzing DNA molecules one at a time has been a tedious process. However, biophysicists from Delft University of Technology and Leiden University have created a method that accelerates the screening of individual DNA molecules by at least a thousand-fold. With this new technology, researchers can now analyze millions of DNA molecules in just a week, as opposed to the years or even decades it previously took. The findings of their study have been detailed in Science.
Studying the way single DNA molecules behave is vital for grasping genetic disorders and creating better medications. Until recently, the examination of these molecules individually was a time-consuming process. A team of biophysicists from Delft University of Technology and Leiden University has developed a method to enhance the screening of individual DNA molecules by over a thousand times. This innovation allows researchers to assess millions of DNA molecules in a week, instead of the years to decades it would have previously required. The results of their research are now published in Science.
“DNA, RNA, and proteins play crucial roles in regulating various processes within our body cells,” explains Leiden Professor John van Noort. “To comprehend the proper and improper functioning of these molecules, we must explore how their 3D structure is influenced by their sequence, which necessitates measuring them one molecule at a time. However, single-molecule measurements can be slow and labor-intensive, especially with the overwhelming number of possible sequence variations.”
From Decades to Days
The research team has created an innovative tool named SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space). This technology enables the simultaneous study of millions of DNA molecules. “Conventional methods that analyze one sequence at a time often require hours of measurement for each sequence. With SPARXS, we can analyze millions of molecules within a single day to a week. Without SPARXS, such analyses would take several years to decades,” states Delft Professor Chirlmin Joo.
“SPARXS allows us to examine extensive sequence libraries, providing fresh insights into how DNA’s structure and function are influenced by sequence. Moreover, this technique can be utilized to swiftly identify optimal sequences for diverse applications, from nanotechnology to personalized medicine,” adds PhD Candidate Carolien Bastiaanssen.
Never Combined Before
To develop the SPARXS approach, the researchers merged two existing technologies that had never been used together before: single-molecule fluorescence and next-generation Illumina sequencing. The first method involves labeling molecules with a fluorescent dye and observing them with a sensitive microscope, while the latter can simultaneously read millions of DNA sequences. Joo explains, “It took us a year to assess whether the combination of these two techniques was practical, followed by four years to create a functional approach and an additional two years to ensure measurement accuracy and consistency while managing the extensive data generated.”
“The real excitement and intrigue began when we needed to interpret the data,” says first author Ivo Severins. “Since these experiments, which integrate single-molecule measurements and sequencing, are completely novel, we had no idea what results we would encounter. It required significant effort to sift through the data to discover correlations and patterns and to understand the underlying mechanisms we observed.”
Overcoming Data Processing Challenges
Another obstacle faced by the team was the management of the enormous data produced, Van Noort notes: “We needed to establish an automated and reliable analysis pipeline. This task was particularly tough because single molecules are delicate and produce only a minimal amount of light, leading to inherently noisy data. Additionally, the data obtained does not straightforwardly reveal how the sequence impacts the structure and dynamics of DNA, even for the relatively simple structures we were examining. To validate our understanding, we developed a model incorporating our DNA structure knowledge and compared it against the experimental data.”
Medical Advances
A more refined approach to manipulating and understanding DNA sequences is likely to result in breakthroughs in medical treatments, including enhanced gene therapies and personalized medicine. The researchers also anticipate biotechnological innovations and a deeper understanding of biological processes at the molecular level. Joo concludes, “We expect that applications in genetic research, drug development, and biotechnology will start to emerge in the next five to ten years.”
