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EU Awards Team $3.3M to Develop Protein Sequencing Platform


NEW YORK (GenomeWeb) – The European Commission has awarded a group of researchers and companies 2.9 million ($3.3 million) to develop new technologies for high-throughput protein sequencing and single-molecule DNA and RNA sequencing.

The project, called "Protein sequencing using optical single-molecule real-time detection" and dubbed PROSEQO for short, commenced last month and will run through February 2019. PROSEQO is being funded through Horizon 2020, the European Framework Programme for Research and Innovation.

Denis Garoli, a researcher at the Italian Institute of Technology in Genoa, is coordinating the project. He told GenomeWeb that PROSEQO plans to develop a device that supports nucleic acid sequencing and protein sequencing using plasmonic nanostructures.

The envisioned technology should improve upon single-molecule sequencing approaches being developed and commercialized by companies such as Oxford Nanopore and Pacific Biosciences by enhancing signal-to-noise ratios during detection and providing "better control of the single-molecule reaction and transit velocity," Garoli said, therefore producing data on par with commercial next-generation sequencing systems, but with the speed and protocols of the emerging platforms.

According to the grant abstract, PROSEQO over the next three years will craft this new sequencing technology relying on plasmonic nanostructures to enhance the optical detection and to control the molecules' movement by means of optical trapping. The project will also use plasmonic-based optical spectroscopy for sequencing proteins and will develop an analytical model to reconstruct sequences from the signals recorded. A plasmonic device that can sequence both nucleic acids and amino acids in one functional unit is also envisioned by the researchers on the project.

"We are introducing, for the first time in sequencing applications, the use of three-dimensional, nano-hollow plasmonic nanostructures," said Garoli of PROSEQO’s concept. He claimed that the use of synthetic structures will permit the concentration of high electric fields in the inner part of the nanochannel. "This strong field confinement allows us not only to greatly enhance the optical signal emitted during the event of biorecognition, but it can also allow a fine control of the molecule translocation through the nanochannel by means of optical trapping," he said.

While some of the work on nanofluidic technology and single-molecule detection has already been carried out at IIT, Garoli said that the project's collaborators are essential to realizing PROSEQO's ambitions. Project members include researchers at Université Paris-Sud and Universitat de Barcelona, as well as industrial partners Berlin-based Alacris Theranostics, and AB Analitica, a Padua, Italy-based molecular diagnostics firm. Garoli is also a researcher at AB Analitica, and he said the company, which will mainly contribute bioinformatics expertise to PROSEQO, will be involved in the potential commercialization of the project's results.

Felix Ritort, head of the Small Biosystems Lab at Universitat de Barcelona, said that his group will contribute to the project a plasmonic optical trap to control the translocation of DNA and proteins, and to detect and discriminate different monomers along the sequence as they translocate through the nanostructures. Initially, Ritort's group will aim to control DNA and protein translocation across a synthetic silicon nitride nanopore.

"This should be useful to quantify under which conditions we can reach translocation speeds for detectable monomer signal and therefore sequencing," Ritort told GenomeWeb.  

Later, the group will rely on a plasmonic trap created by illuminating the nanostructure and forming a non-radiative electromagnetic nanotrap that is capable of slowing down the speed of the translocating polymer, Ritort noted. "The aim of the plasmonic trap is to provide sufficient signal-to-noise ratio for fluorescence detection of the different monomers as they go translocate," he said.

Niko Hildebrandt, head of the Nano Bio Photonics group at Université Paris-Sud is involved in providing PROSEQO with tools for optical spectroscopy. Hildebrandt told GenomeWeb that his group is developing Förster resonance energy transfer (FRET) biosensors, which employ pairs of fluorophores to detect "very tiny distance modifications," such as when a DNA strand or a protein strand is passing in between them.

"We want to design FRET sensors that provide a base or amino acid-specific FRET signal," said Hildebrandt, thus providing the core detection method for the planned device.

Along with AB Analitica, Alacris will provide bioinformatics assistance to PROSEQO. CEO Bodo Lange told GenomeWeb that Alacris will help to define new algorithms for data analysis and sequence reconstruction as well as "testing the performance of new sequencing methodologies against the current state of the art."

Alacris combines its molecular analysis and bioinformatics capabilities in a product called ModCell, a "systems medicine modelling approach" for matching patients with therapies. The company also offers sequencing services and is an Illumina certified service provider.

Lange said Alacris' involvement in PROSEQO "aligns well" with the company's mission to aid in the development of new sequencing technologies that will "drive the field of personalized medicine forward" and "provide a more efficient path from patient sample to accurate and reliable model-based predictions."

Garoli called protein sequencing the "real high-risk, high-impact goal" of the project. If the direct sequencing of single proteins is realized, the proposed technology could have a "tremendous impact on the future of personalized medicine," Garoli said, allowing for more precise monitoring of disease response to therapeutics at the molecular level.

Proteins can yield "far more compelling revelations than may be gleaned from DNA alone," the investigators noted in the grant abstract. Single-molecule sequencing of proteins is of "enormous value," they continued, "offering the potential to detect diminishingly small quantities of proteins that may have been altered by alternative splicing or post-translational modification."

Still, Garoli acknowledged that while the proposed technologies have the potential to make "stunning advances" possible for human health, protein sequencing remains difficult, hampered by the fact that proteins cannot be amplified, have to be unfolded and often fragmented, and that in contrast to DNA, fluorescent amino acid tags without spectral overlap do not exist.

Given these challenges, Garoli conceded that the full development of the technology will require more than the three years covered in the grant.

"The aim of the project is to demonstrate the feasibility of the idea," said Garoli. "After that, our final goal will be the complete development of a prototype and ... possible commercialization that can be done in collaboration with our industrial partners."