NEW YORK (GenomeWeb) – Scientists from Rice University have found a way to make DNA hybridization more specific and have formed a startup firm to pursue commercial applications, such as the detection of low concentrations of single nucleotide variants (SNVs) found in circulating cell-free tumor DNA.
Juexiao Sherry Wang and David Yu Zhang, scientists at Rice University and co-founders of the startup firm Searna, detailed their two-step approach to increasing hybridization specificity in a paper published last week in Nature Chemistry. The method started with a theoretical analysis of the thermodynamic properties of DNA binding to inform the sequence design of probes to be used experimentally. It includes a new algorithm to optimize the kinetic parameters of DNA hybridization and a specially designed oligonucleotide probe to enrich the target sequence.
The algorithm determines the optimal sequence design for two different molecules, a probe that binds to an SNV, like a cancer mutation, and a molecule called a "sink" that binds to the corresponding wild-type sequence, which could represent healthy DNA. When designed properly, the sink removes the wild-type DNA from solution. "We're enriching the sample for the target sequence that we're looking for," Zhang said.
The probe then combines the oligos binding to the SNV and the wild-type DNA into a single probe. Using this approach, Zhang said they were able to detect rare mutations at less than 1 part in 1,000. That's 30 times better than existing methods to improve hybridization specificity, he said, and on par with optimized PCR assays.
Their method has formed the basis for Searna, a startup that hasn't settled on where to do business, but has already begun work with several partners throughout the molecular biotechnology industry. "It's so universal that it could be useful for a lot of applications." The firm has already submitted five patent applications addressing different aspects of the technology, from the four-stranded probe architecture, to uses in PCR and next-generation sequencing target enrichment, to algorithmic sequence design.
Zhang used the classic "needle-in-a-haystack" metaphor to describe detecting SNVs among other DNA. He described two approaches to find such a needle, both of which are illustrated in the Nature Chemistry paper: using a magnet to stick to the needle and pull it out of the hay and using a herd of goats to eat the hay and make a much smaller haystack. The algorithm to design the probe makes for a better magnet, he said, while the "sink" molecule designed to bind to the wild-type DNA acts like the goats, reducing the amount of hay to look through.
The software is the key to getting the specificity, Zhang said, simulating the hybridization process to yield an optimal probe and sink design."There's no way to analytically solve for the set of optimal conditions for every single probe. You have to do it in silico."
Zhang considers the algorithm so crucial to Searna's success that it has not been made publically available. It's an extra step in the workflow, but it takes less than an hour to run the software and give the optimal oligo design for a given target sequence, he said.
Zhang said that Searna can get the system to work with any downstream monitoring technology and that he is currently working on NGS sequencing readouts. "Hybrid capture for NGS target enrichment is something we're heavily investing in right now," said Zhang.
The technology's ability to enrich for rare mutations could be useful in a number of other scenarios as well. Zhang listed cancer mutation detection in PCR, cell-free DNA and circulating tumor DNA in liquid biopsies, and detecting fungal DNA in sepsis as possible use cases for Searna's technology.
Between the software algorithm and the probe design, Searna has started a number of collaborations with different firms including DNA manufacturer Integrated DNA Technologies, the Austrian RNA-seq firm Lexogen, BGI, and NanoString, Zhang said. He also noted that PCR is an attractive area for collaboration because Searna's method works on optimizing hybridization, while PCR companies were improving specificity by using better reagents and chemistry. "We can tackle the problem from two sides by pulling enzymes and digital PCR in," Zhang said. He added that Searna is working with a large PCR-based infectious disease diagnostics company, but declined to disclose which one.
In the meantime, Zhang is looking to prove how the DNA hybridization probes could prove useful in clinical studies. "We're trying to look for very early-stage cancer patients who have rare DNA in their blood," he said.