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Catalyst-Based Thermodynamics Method Could Enable Improvements in DNA Oligo Design

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NEW YORK (GenomeWeb) – Researchers at Rice University have developed a catalyst-based method for determining melting properties of oligonucleotides.

The method, published last week in Nature Communications, can be used to more accurately determine DNA hybridization thermodynamics, and may enable improvements to oligonucleotide construction for applications such as making PCR primers.

The basic biophysics of how strong or weak a given oligonucleotide interaction will be is very poorly understood, David Zhang, a professor of bioengineering at Rice and corresponding author on the study, told GenomeWeb. "If you ask anyone who has done PCR primer design, you have to basically go through several rounds of trial and error," he said.

A primer that's too strong winds up binding non-specifically everywhere, while a primer that is too weak tends to not bind at all, resulting in no amplification — there’s a "fine Goldilocks zone in which you can actually get good behavior," Zhang said. Ultimately, the problem stems from sets of computations that "people started working on maybe 40 years ago, and stopped working on maybe 25 years ago," he explained.

At the melting temperature of a particular DNA structure it would be exactly half bound and half unbound, but information on how strong or weak binding is at all other temperatures must be extrapolated from the melt temp data point. Zhang's method, on the other hand, looks at DNA binding "in its native conditions," and also zooms in to specific motifs to enable predictive design of oligos, he said.

While multiple iterations of primer design may be worth the cost in a clinical assay, time and R&D costs need to be kept low for most researchers. "You want to design primers and probes so that they work well the first time," Zhang said, adding that "better knowledge of how the DNA behaves and interacts with other molecules is important for that."

Michael Zuker, a professor at Rensselaer Polytechnic Institute, told GenomeWeb that this study should prove quite valuable to those in the nucleic acids field.

Zuker has developed an algorithm called FASTH that searches RNA or DNA sequence databases for optimal hybridization sites for nucleic acid query sequences, using hybridization free energy as the criterion for selection. The Nature Communications study is "exciting," and "a major breakthrough in measuring energy parameters for DNA hybridization," Zuker said, noting that it could make software to design custom DNA oligomers, such as FASTH, even more useful.

Zhang's method uses an unconventional catalyst to speed up the equilibration reaction that is ultimately used to infer thermodynamic properties — a reaction which can sometimes take several months — by five orders of magnitude, Zhang said. The catalyst is a "rationally designed" sequence of DNA that differs for each target sequence.

"We have a bioinformatics pipeline to figure out how to design this catalyst, it's a unique molecule for each thing you want to characterize," he said.

The method can also be used to determine the ways different oligo parameters, like SNPs or single-base bulges, affect thermodynamic properties. The Nature Communications study, for example, specifically assessed a parameter called a dangle. Zhang hopes to assemble a database of measurements that show how every individual parameter can affect an oligo's melt temperature.

"It's kind of this dirty little secret ... but there are many parameters that just aren't well characterized," Zhang said. "There are a lot more parameters that need to be done, and we hope that other labs will help collect the data."

Although Zhang's lab is pursuing IP on its other biotechnology work to look at rare, single base changes — for example, a probe design approach using simulations of binding kinetics, and "continuously tunable" probes — Zhang does not intend to patent the catalyst-based technique. Instead, he envisions it being used in a "worldwide team effort to get nucleic acids up to modern times, rather than being 30 years in the past."

Applications could also include probe-based capture of genomic DNA sequences for analysis, for which designers must currently take a more empirical, "shotgun" approach, Zhang added. "My hope is that all nucleic acid sciences can become much more knowledge-driven. Unlike proteins, the tools basically don't exist for very good prediction of structure and binding strength for nucleic acids [but now] we have the capability to be much more rational design- and knowledge-driven." 

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