Describe the kinetics of DNA hybridization

Nanoscientists and theoretical physicists at EMBL Australia’s Node in Single Molecule Science UNSW Medicine & Health have teamed up to uncover the intricate mechanisms that govern how quickly two matched DNA strands can fully unite – or hybridize – to form double-stranded DNA. Their findings were published in the journal Nucleic Acids Research.

A theory was put forward about 50 years ago that hypothesized that how fast a DNA strand hybridizes is determined by the initial contact that leads to further binding of the matching base strand to the DNA strand – called nucleation interactions. Until now, this theory has never been proven due to the many complexities surrounding DNA biology.

“There are a large number of pathways by which two completely separate strands can bind to each other. Standing DNA doesn’t come together into a fully hybridized duplex in an instant. At some point, only two or three base pairs will spontaneously combine. This is a nucleation event,” said Associate Professor Lawrence Lee, who led the research team from UNSW Medicine & Health, UNSW Science and Imperial College London.

“We built a simple mathematical model, which has only two parameters, and asked: if we only knew how many nucleation interactions there are, and how stable they are, can we predict the degree of hybridization? And we found that the answer is yes,” he said.

To test this model quantitatively, the research team translated the original hypothesis into a mathematical formula they could use to measure their experimental observations with synthetic DNA.

A/Prof Lee explains that simplicity is critical to the predictive power of their model.

“If a mathematical model contains too many different parameters, it is no longer useful for making predictions. The main difference from previous attempts to understand the degree of DNA hybridization is that our model has multiple parameters and is tested against DNA sequences that should not form secondary structures,” he said.

The secondary structure of DNA is formed when the strand folds onto itself, potentially obscuring the nucleation and binding sites.

“The theory is, if this initial small interaction is stable enough, it will move from there to the very fast zipper of the DNA strand. If the limiting step is nucleation, then if you have more nucleation states, the DNA will hybridize more quickly,” said A/Prof Lee.

This discovery has the potential to improve our understanding of biological systems. The ability to predict or control the rate of DNA hybridization could also help refine or expand the usefulness of nanotechnology. With this new understanding, the researchers were able to adjust the number and stability of the nucleation interactions and, in turn, control the rate of DNA binding. This can be achieved in many ways, including by changing the reaction temperature, DNA sequence, and the ionic strength of the solution.

“We were able to produce high-resolution images using DNA paint – fluorescent strands of DNA used as tags for the microscope – because we measured the binding and release of DNA to individual molecules. But, it takes a long time to get the data. If we can rationally design the sequence for DNA paint, so that it can bind faster, then we can reduce the acquisition time for super-resolution imaging,” said A/Prof Lee.

Reference:

1. Sophie Hertel, Richard E Spinney, Stephanie Y Xu, Thomas E Ouldridge, Richard G Morris, Lawrence K Lee. Stability and number of nucleation interactions determine the degree of DNA hybridization in the absence of secondary structures. Nucleic Acid Research, 2022; DOI: 10.1093/nar/gkac590

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