PROtein SEQuencing using Optical single molecule real-time detection

PROSEQO project will pave the way for the development of new generations of sequencing technologies, both for proteins and nucleic acids. Specifically, we will develop a complete and entirely new technology and tool for accurate and low cost NGS. A new technology to provide enhanced optical detection will be developed. Revolutionary new optical and molecular designs will be developed, such as novel nanostructures, FRET intensity and lifetime foot printing, and optical traps for biomolecules translocation, to achieve single molecule sequencing (SMS) and to go a significant step beyond the state-of-the-art of single molecule protein (and DNA) sequencing. We will test our technology starting from a few well-defined molecules up to an integrated system that will also allow high quality reconstruction of the sequence by means of data analysis.
The highly interdisciplinary effort associated with PROSEQO will lead to:
· development of advanced optical technologies based on new plasmonics devices
· highly engineered systems for molecular translocation / movement control
· new molecular designs for enhanced FRET spectroscopy rulers
· integration of the above technologies within a core multifunctional platform and
· iterative refinement of the data analysis based on assessment of instrument performance in SMS, both of nucleic acids
and amino acids.
Long term vision: the technological advances brought about by PROSEQO will:
· make available novel technologies for third generation sequencing of protein and DNA/RNA
· contribute to our understanding of complex biological networks
· significantly advance proteomics research and biomarker development
· facilitate the development of new tools to characterize heterogeneous molecular ensembles through accurate and fast
sequencing

In order to achieve the final goal of the project a step-by-step approach is followed. During the first year all the preliminary aspects regarding design, theoretical investigation, nanofabrication, chemical functionalization and optical setup have been faced. In particular, as defined in the Grant Agreement, in IIT the research work has been focused on optimization of plasmonic nanostructures characterized by a 3D design comprising a axial nanochannel. These structures ensure the localization of the enhanced electric field at the desiderated point where the single-molecule is going to be detected. Both theoretical design (deliverable 2.1) and nanofabrication procedures (deliverable 2.2) have been investigated and several results have been achieved. Moreover, as defined in the proposal, in IIT a complex computer simulation (deliverable 3.1) has been setup in order to investigate, at molecular dynamics level, the interaction between FRET couples and a biomolecule (in particular nucleic acids and amino acids chains). Also during this first twelve months, the other scientific partners, i.e. UBE and UPSud, started the investigation on nanostructure functionalization with FRET dyes (deliverable 3.3) and on optical setup for single molecule optical trapping (deliverable 4.1). Both of these activities need more time to be optimized and finalized for the project porpoise. Finally AbA worked on the definition of how the biological samples will be treated (deliverables 5.1 and 5.11).
The work performed during this first period represent valuable results for the activities started for the second year. From now on, single molecule translocation and detection experiments will be setup by using the nanostructures realized during this period. Moreover, once the most suitable FRET strategy will be defined, the functionalization strategy defined during the first year will be exploited for optical spectroscopic detection of single molecules. The movement of these latter will be controlled by means of optical trapping thanks to the work now ongoing in UBE.

To date, few studies have focused on developing new technologies for single molecule protein sequencing The large number of amino acids to be discriminated (up to 20) requires the development of a completely new detection solution, able to feature unambiguous discrimination between the different target molecules at extremely high detection sensitivity. Plasmonic enhanced optical spectroscopy will be exploited in order to distinguish the single amino acids, exploiting the effects offered by plasmonics to obtain a high SNR. The possibility to design plasmonic nanotraps of sufficiently high stiffness will lead to increased SNR as compared to standard optical trapping approaches.
The expected impact of PROSEQO is to initiate a radically new line of technologies by establishing the proof-of-concept of a NGS methodology.
PROSEQO constitutes an early stage, high risk paradigm of visionary science and is based upon the collaborative research efforts of different participants, highly competent in their fields and motivated to a successful exploration of new founding principles.
The novel multi and interdisciplinary approach of PROSEQO has a large potential to lead to groundbreaking changes in complete proteome mapping, which will provide enormous benefits to the healthcare sector. Indeed, the early stage identification of a disease-specific marker can greatly improve the clinical success rate and reduce mortality, notably in proteinprone diseases such as neurodegenerative and protein-aggregation diseases

Periodic Reporting for period 2 - PROSEQO (PROtein SEQuencing using Optical single molecule real-time detection)

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