Researchers at Northwestern University found that DNA strand separation may require more force than previously thought when modeled in a more true-to-life environment.
In most labs, scientists studying DNA place it into a simple, water-based solution. This controlled setup lets researchers handle DNA without interference from other molecules. To separate DNA strands, these labs often heat samples to temperatures above 150 degrees Fahrenheit, a temperature a cell would never naturally reach.
In contrast, DNA within a living cell exists in a highly crowded environment, where specialized proteins bind to the DNA, mechanically unwinding and separating the double helix.
“The interior of the cell is super crowded with molecules, and most biochemistry experiments are super uncrowded,” said Northwestern professor John Marko. “You can think of extra molecules as billiard balls. They’re pounding against the DNA double helix and keeping it from opening.”
Marko, who teaches molecular biosciences and physics at Northwestern’s Weinberg College of Arts and Sciences, collaborated closely with post-doctoral researcher Parth Desai.
Their team used advanced microscopic tools called magnetic tweezers. With these tools, they carefully attached tiny magnetic particles to DNA strands secured at one end, gently pulling the strands apart and capturing the process using precise imaging. Marko was among the first scientists to theorize and use this innovative method, which has existed for 25 years.
What they found
Marko and Desai wrote a paper, published in the Biophysical Journal, that not only identifies but also quantifies the amount of stress imposed by crowding.
Desai introduced three types of molecules to the solution holding DNA to mimic proteins and investigated interactions among glycerol, ethylene glycol, and polyethylene glycol (each approximately the size of one DNA double helix).
“We wanted to have a wide variety of molecules where some cause dehydration, destabilizing DNA mechanically, and then others that stabilize DNA,” Desai said. “It’s not exactly analogous to things found in cells, but you could imagine that other competing proteins in cells will have a similar effect. If they’re competing for water, for instance, they would dehydrate DNA, and if they’re not competing for water, they would crowd the DNA and have this entropic effect.”
What’s next
While fundamental, research like this has “been the basis for many, many, many medical advances,” Marko said, such as deep sequencing of DNA, where scientists can now sequence an entire human genome in under a day. He also thinks their findings may be broadly applicable to other elements of fundamental biochemical processes.
“If this affects DNA strand separation, all protein interactions with DNA are also going to be affected,” Marko said. “For example, the tendency for proteins to stick to specific sites on DNA and to control specific processes — this is also going to be altered by crowding.”
In addition to running more experiments that incorporate multiple crowding agents, the team hopes to move closer to a true representation of a cell, and from there, study how interactions between enzymes and DNA are impacted by crowding.
Reference: “Molecular crowding suppresses mechanical stress-driven DNA strand separation” by Parth Rakesh Desai and John F. Marko, 30 April 2025, Biophysical Journal.
DOI: 10.1016/j.bpj.2025.04.024
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