Before they can design diagnostic reagents that can recognize specific bits of DNA and RNA, scientists typically resort to a kind of tracking. While they stalk their quarry, scientists pick up thermodynamic signs, such as experimental melting temperature and free energy values, that they hope will point them in the right direction. They extrapolate from these laboratory-derived signs to guess at the values their DNA and RNA quarry will have “in the wild.”
Unfortunately, conditions in laboratory investigations and those in the wild can be quite different, leading scientists astray. To narrow the difference between laboratory and native conditions—and prevent error-prone extrapolations—scientists at Rice University have developed a way to measure exact thermodynamics at the temperatures and other conditions that are more physiologically relevant. As Rice bioengineer David Zhang, Ph.D., puts it, “We are studying DNA ‘in the wild’ rather than observing DNA molecules that have been locked up in an overheated cage.”
Animals in the wild are hard to study because they can take a long time to come into a camera's field of view. The same observational challenge applies to the study of DNA in its native conditions.
“Typically, it would take months of observing until a researcher is able to find the true state of the DNA, and that's too long to wait,” said Dr. Zhang. “We have developed a system that helps us quickly zoom in on the DNA molecules being studied. Typically, because we are speeding the reaction up 10,000- or even 100,000-fold, we only have to wait a couple of hours to do our analysis.”
Dr. Zhang noted that while the new technique is more labor-intensive than a melting curve, it generates far more accurate results, as proven by the hundreds of molecules his lab has already analyzed. The errors that the Zhang research team has encountered are roughly tenfold lower than previous methods; this allows for more accurate rational design of DNA diagnostic reagents. A high-resolution melt experiment would have to be repeated dozens of times to generate an average result with close to the same precision, he said, but even that would not mitigate errors from testing under unrealistic conditions.
Details of the new technique appeared January 19 in the journal Nature Communications, in an article entitled, “Native characterization of nucleic acid motif thermodynamics via non-covalent catalysis.”
“The equilibrium constant of a reaction with thermodynamics closely approximating that of a desired motif is numerically calculated from directly observed reactant and product equilibrium concentrations; a DNA catalyst is designed to accelerate equilibration,” wrote the authors. “We measured the ΔG° of terminal fluorophores, single-nucleotide dangles, and multinucleotide dangles in temperatures ranging from 10 to 45°C.”
Typically, to study the behavior of a particular DNA sequence—for example, one that is specific to a virus—researchers would heat and cool the molecules to observe their fluorescence at different temperatures. Using rules of thumb, researchers would then guess the DNA's properties at temperatures other than the one measured. However, such approaches are inaccurate because the way a DNA molecule behaves at 75°C is a poor predictor of how it behaves at 37°C.
“Our goal is to build a database of good DNA and RNA thermodynamic parameters. Melt curves done in the '80s and '90s are too crude,” Dr. Zhang explained. “Unfortunately, that's what people in diagnostic and life sciences research use today because there's been no better method.
The Rice method is the first for which the Zhang lab will not seek a patent. “This is something I feel is beneficial to the entire world,” he declared. “I want people to use this so we can build thermodynamic databases together. We've basically started the process of forming the database, but we're nowhere near the end.”