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PicoSeq Expanding Potential Applications of Hairpin DNA-based Technology As It Enters First Financing Round

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Paris-based startup PicoSeq has started its first major financing round — the latest step in the firm's effort to automate and commercialize its mechanical, DNA hairpin-based method for assessing single molecules of DNA.

The company is currently seeking funds from a variety of sources and/or potential partners, PicoSeq CEO Gordon Hamilton said, and hopes to reach its current target of roughly €5 million ($6.6 million) late this spring or sometime over the summer.

"It's a relatively large, reasonably substantial first round, but we consider PicoSeq as being a development-phase business," Hamilton told In Sequence.

"Essentially, the major research questions and research problems have been solved," he added. "So it's a matter of taking what we have and then scaling it to being commercially useful."

While the overall strategy used to interrogate individual pieces of DNA has not changed significantly from that described in a proof-of-principle study in Nature Methods last spring (IS 3/13/2012), Hamilton explained that the past year has seen research developments related to the types of information that can be gleaned from each DNA molecule.

In particular, those developing the mechanical DNA assessment approach have come up with a strategy for looking not only at the sequence of individual DNA molecules but also at modifications affecting nucleotides within that molecule.

With such research underway, PicoSeq ultimately aims to produce a commercial instrument capable of everything from DNA fingerprinting and detection of epigenetic modifications to full sequencing.

The basic technology behind such a proposed system centers on a DNA hairpin with one end attached to a magnetic bead and the other end tethered to a solid surface. Applying a magnetic force to the system opens the hairpin so that it can interact with corresponding oligonucleotides present in the surrounding solution.

When interactions between the hairpin and oligos occur, they prevent the hairpin from fully re-closing. And the position of the magnetic bead — relative to where it would be in a fully open or closed hairpin — provides information on where in the DNA hairpin the blockage occurred, providing sequence information.

"Conceptually, one can think of that as being a little bit like an inverted microarray," Hamilton said, "except that rather than fixing the probes to the floor of a flow cell you're fixing the DNA to the floor of the flow cell and then washing with probes."

Hamilton credits Laboratoire de Physique Statistique, Ecole Normale Supérieure researchers Vincent Croquette, David Bensimon, and Jean-François Allemand with developing the technology as it stands today. While the researchers are not expected to join PicoSeq full-time, he noted that all three will serve as scientific advisors to the startup.

"We are working very, very closely with the original research lab where the intellectual property was generated and created," Hamilton said, "and where the magnetic tweezer instrument was developed over the last decade or so."

For the time being, most of the basic research activities are continuing to take place in that lab, though much of the research and potential product development is expected to shift over to PicoSeq proper once the current funding round is completed.

Though PicoSeq remains something of a shell company at present, the firm has "really taken shape" over the past year, according to Hamilton, who noted that progress has been made in fleshing out the management team, in particular. That group now includes Chief Technology Officer Chas André as well as several senior scientists or scientific advisors.

On the research front, meanwhile, the group has been working to broaden the types of information that can be gleaned from each of the DNA molecules, with an eye to extending the potential applications for the technology.

Along with using oligos to determine the sequence of DNA or identify sequences containing a specific DNA barcode of interest, the researchers are finding ways to see DNA modifications on these molecules as well — work that Hamilton presented at the annual Advances in Genome Biology and Technology meeting at Marco Island, Fla., earlier this year.

Rather than relying exclusively on oligos to bind DNA and prevent the hairpin from reforming, he explained, the team has shown that it can produce hairpin blocks with antibodies as well, making it possible to see specific DNA modifications.

"Our ability to detect DNA modifications uses exactly the same principle [as that described previously] and, in fact, can use exactly the same fragments of DNA," Hamilton said.

"Instead of flowing into the flow cell an oligonucleotide of known sequence in order to liberate sequence information, we instead flow in an antibody — or in other forms that might be an aptamer or other molecule — that is generated against a particular base modification," he said.

Again, by releasing magnetic force on the hairpin during the time frame that a given antibody is bound — determined from that antibody's binding kinetics — researchers can use the magnetic bead position to detect when and where an antibody is interacting with the DNA molecule in a hairpin.

So far the group has demonstrated the feasibility of detecting 5'-methylcytosine marks in this manner, Hamilton explained, though the approach is expected to be applicable to any base modification for which an appropriate interacting probe exists.

"There is nothing to say that the principle could not be applied against any modification for which you could conceivably generate an antibody," he said, noting that researchers are already starting to test the same approach on other types of modifications.

Another area of ongoing interest for researchers — and something that PicoSeq plans to pursue even more vigorously once it secures its initial funding — is finding ways to boost automation of the hairpin DNA-based method.

"The foundations [of the technology] are now very strong and the proof-of-concept data is very robust," Hamilton said. "But [with] the instrument in its current form, we would never be able to do things at the sort of scale that would be required in order to be commercially viable for sequencing-related activities."

Nevertheless, he noted that the company has a "very aggressive timeline" for generating commercial-grade proof-of-concept data and producing a commercial-grade instrument.

Hamilton did not disclose whether that instrument would be more apt to rely on a hybridization or sequencing-by-ligation-based scheme, but he said that the researchers are "trying to work out what the best strategy would be for our full sequencing approach."

The anticipated price of such a machine is yet to be determined. But Hamilton noted that the hairpin DNA-based setup being used in the lab is simple, compact, and somewhat akin to a modified light microscope.

"There are no components within the instrument that are in any way hugely technically sophisticated, complicated, or expensive," he said. "It is a very simple and very elegant approach."

As the company gears up for future commercialization efforts, another area being given more attention is sample preparation. Ideally, Hamilton explained, sample preparation procedures for the PicoSeq instrument would be on par with the sorts of sample preparation protocols already being used for DNA sequencing.

Even so, Hamilton emphasized that he does not see PicoSeq as a direct competitor to existing or anticipated next-generation sequencing instruments. Rather, he argued that the mechanical hairpin method might have a range of applications that are distinct from — or complementary to — those offered by such platforms.

For instance, because each DNA hairpin can be opened and closed tens of thousands of times, it should be possible to look at the same molecule multiple times — perhaps searching for a DNA fingerprint or barcode in one instance and profiling a full sequence or a suite of specific epigenetic modifications in another.

"You could potentially build up the level of detail that you want from the picture based on your requirements as a user," Hamilton said, "rather than taking the kind of sequencing approach where you chuck everything in and sequence the whole lot and deal with the data afterwards."

The nature of the technology also allows for distance measurements between probes — something Hamilton sees as a potential advantage in situations where researchers or clinicians are interested in determining the space between bits of sequence, such as the ends of a clinically informative repeat. As such, he said, the technology could eventually find its way into not only basic and applied research but also into more clinical settings.

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