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Polyplus-transfection Demonstrates Use of ZNAs as Short Hydrolysis Probes for qPCR

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By Bernadette Toner

Polyplus-transfection has demonstrated that its proprietary zip nucleic acids, which are modified oligonucleotides that have a greater affinity for nucleic acids than standard oligos, can be used as short hydrolysis probes for qPCR.

The work follows on a study the Strasbourg, France-based developer of nucleic acid-delivery technology published last year that showed that ZNAs could serve as effective primers for PCR and RT-qPCR, with some "distinct advantages" over unconjugated DNA primers and locked nucleic acid primers.

In a new paper published last week in Nucleic Acids Research, company scientists showed that the molecules also work as hydrolysis probes in quantitative PCR.

ZNAs are oligos that have been modified with conjugated spermine derivatives, called Z units, that decrease electrostatic repulsion and improve hybridization. The company claims that ZNA enables nucleic acid strands to join together much like a zipper, a characteristic that lent the technology its name.

The melting temperature of the ZNA increases linearly with the number of Z units grafted onto an oligo, according to the firm, so ZNAs can easily be fine-tuned for specific hybridization temperatures.

Last November, the company signed a non-exclusive licensing agreement with Sigma-Aldrich that gave the biochemical supplier worldwide commercialization rights to the ZNA technology.

Leila Guerlain, who leads marketing and business development for the ZNA product line at Polyplus-transfection, told PCR Insider this week that Sigma plans to sell ZNAs for use as primers, probes, and other nucleic-acid applications in the research market.

Guerlain said that ZNAs would be suitable for nearly any nucleic acid technology, including in situ hybridization or microarrays, though she acknowledged that "further development would be necessary to use ZNAs in such applications."

She said that the company decided to develop ZNAs for PCR applications first "in order to show off the properties of these kinds of oligonucleotides to hybridize with their targets."

But the molecules demonstrate "very interesting properties that can also be applied in all nucleic acid-based technologies … and many applications other than PCR," she added.

Stacey Hoge, product manager for custom DNA and PEPscreen products at Sigma-Aldrich, said that the company expects the ZNAs to be available before the end of the quarter, and that the first application will be as dual-labeled probes for qPCR applications.

Hoge added that the company is working with several researchers on developing additional applications for the technology.

She said that the company has not yet set pricing for the ZNA molecules, but is willing to provide the technology for interested researchers on a "quotation basis."

'Potent Probes'

In its previous study on the use of ZNAs as primers, Polylus-transfection demonstrated that ZNAs work at higher annealing temperature, low primer concentrations, lower magnesium concentrations, and much quicker than unmodified oligonucleotides or locked nucleic acids.

ZNAs were also shown to improve the yield of cDNA synthesis in reverse transcription when compared to current methods, and they were able to detect genomic targets at 10-fold lower concentrations than DNA- or LNA-substituted primers.

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Given those findings, "it was quite obvious that this kind of molecule could be valuable as probes as well," said Nathalie Lenne, a project leader at Polyplus-transfection and the corresponding author on both studies. "It was quite straightforward to try."

Hydrolysis probes, which contain a fluorophore and quencher at either end, are particularly tricky because the length of the probe can affect quenching efficiency and background fluorescence. While shorter probes are desirable for increasing target discrimination, shorter probe length can decrease the melting temperature, which can negatively impact probe binding during PCR cycling.

Given the ability of ZNAs to increase the melting temperature in a controlled manner, Polyplus decided to look into their applicability as short hydrolysis probes.

In the NAR study published last week, the authors compare the performance of ZNA hydrolysis probes with unmodified DNA probes, probes conjugated with minor groove binders, and probes containing locked nucleic acid probes.

In the comparison of a 17-mer ZNA probe to a standard DNA probe, "the ZNA probe performed much the same as a longer standard probe containing 22 bases for detection of the wild-type target … [but] due to its shorter length, the ZNA probe showed significantly improved SNP discrimination," the authors wrote.

When it came to mismatch discrimination, the authors found that MGB modification reduced the melting temperature except when the mismatch was within the MGB-binding region — a finding that "confers a strong advantage to ZNAs, making probe design much easier."

While ZNAs did not discriminate mismatches any better than LNAs, the authors note that "LNA-containing probe design remains difficult due to [melting temperature] variation with sequence, position, and number of modifications."

The authors conclude that ZNA probes "are potent short hydrolysis probes," though they concede that "more data have to be generated to determine the rules that will define the number of cationic moieties required for a given sequence."

Lenne said that in addition to this work, the company is also working on non-hydrolysis probes for PCR, and has seen "promising results" so far.

She said that the company is also looking into the use of ZNAs with siRNAs to help improve the delivery of those molecules to cells without the use of transfection reagents — work that is more in line with the company's specialty in transfection technology.

"We want also to continue to develop ZNA oligos for PCR and nucleic acid-based technology," Guerlain said. She noted that the company would like to sign additional license agreements similar to the one that it has with Sigma-Aldrich "to make ZNAs available as custom oligos for R&D purposes."

In addition, the company is also looking into commercializing ZNAs as custom oligos for diagnostic applications.

"We would like to work with diagnostic companies to help them integrate ZNA into commercial kits for IVD purposes," Guerlain said, though she stressed that the molecules are not yet available for diagnostic purposes.

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