Project

Abstract

The advent of analytical techniques with extremely low limits of detection has led to dramatic progresses in the field of nucleic acids sequencing. Despite the development of next generation sequencing platforms, the current genome sequencing task remains formidable, and revolutionary advances in DNA sequencing technology are still demanded. Another technological revolution would be a fast and reliable sequencing of proteins, which are the primary actors in virtually all life processes and are coded by DNA sequences known as genes. Proteins can yield far more compelling revelations than may be gleaned from DNA alone. Protein sequencing may radically transform patient treatment by enabling precise monitoring of disease response to therapeutics at the molecular level. Single-molecule sequencing of proteins would be of enormous value by offering the potential to detect extremely small quantities of proteins that may have been altered by alternative splicing or post-translational modification. In this project, we build upon current state-of-the-art sequencing technologies to develop novel proof-of-principle technologies for high-throughput protein sequencing and single molecule DNA/RNA sequencing. The work proposed herein will provide: (i) a new sequencing technology that utilizes plasmonic nanostructures to enhance the optical detection and to control the molecules movement by means of optical trapping; (ii) a novel approach of plasmonic enhanced FRET and SERS spectroscopy for sequencing of proteins; (iii) a rigorous analytical model to reconstruct the exact sequence from the signals recorded; and (iv) a single molecule sequencing device that can perform sequencing of both nucleic acids and amino-acids in one functional unit. The results of the project will provide a foundation for the use of novel technologies in a wide range of applications, such as next generation protein sequencing, advanced high-throughput DNA sequencing, biomarker discovery, and genetic diagnostics.

The Project in Brief

The main scientific breakthrough of PROSEQO will be:

the development of a novel plasmonic platform for next-generation single protein sequencing. The proposed platform will be developed for the sequencing of long chains of amino acids (proteins). The development of single molecule protein sequencing will be a paradigm change for proteomics, an area in which current sequencing techniques appear limited by low throughput and high costs and a current lack of single molecule approaches. The proposed technology will also be applicable to nucleic acid sequencing (both DNA and RNA), with the development of a significantly advanced single-molecule DNA/RNA sequencing approach being the minimum outcome of the project.
The expected breakthrough of PROSEQO will contribute:

to the development of new technologies for biomolecule detection, trapping and sequencing. In particular, while single-molecule DNA sequencing technologies already exist but can still be significantly improved and optimized, next generation protein sequencing is yet to come. The development of single-molecule protein sequencing will be a milestone for proteomics, e.g. in the search for next generation protein biomarkers, and it has the potential to radically transform patient treatment toward efficient personalized medicine approaches.
The general objective of PROSEQO is the development:

of a proof-of-concept platform for fast and inexpensive sequencing of both proteins and DNA / RNA. The PROSEQO approach will sequentially and directly identify biomolecules sequences in very long fragments from a molecule-dependent optical signal produced by plasmonic nanostructures characterized by two important aspects (Fig.1, Fig.2): A) 3D nano-hollow structures able to channel the movement of the molecule; B) engineered electromagnetic field distribution able to generate giant field localization for both enhanced optical spectroscopy and optical trapping. The final system is intended to provide high quality sequencing of proteins, DNA and RNA without amplification and with minimal preparative steps.

Here we are introducing, for the first time in sequencing applications, the use of 3D nano-hollow plasmonic nanostructures. They can ensure a high degree of freedom in electromagnetic field engineering (localization and distribution) and, with respect to a synthetic nanopore or a planar plasmonic structure, will permit the concentration of very high electric fields (giant fields – up to 102) in the inner part of a nanochannel. This strong field confinement allows us not only to greatly enhance the optical signal emitted during the event of biorecognition (with a consequent higher signal-to-noise ratio - SNR), but it can also allow a fine control of the molecule translocation through the nano-channel by means of optical trapping. PROSEQO will take advantage of these characteristic properties and will combine them with biochemistry / biotechnology methods in order to detect and discriminate single amino-acids. The large number of amino acids to be discriminated (up to 20) requires the development of a completely new detection solution. A radically new approach for plasmon-enhanced fluorescence of translocating biomolecules based on Förster Resonance Energy Transfer (FRET) multiplexing lifetime and intensity foot printing will be used to detect single biomolecules once they pass over the giant-field zone. This new approach will be based on a single color detection leading to an important simplification of the current FRET signal used for sequencing. A single color approach will lead to less complexity in the opto-electronics required for optical detection and storing, and it will also reduce the complexity of the data to be analyzed for sequence re-alignment.

The main characteristic of PROSEQO is the strong interconnection between different technological and scientific fields. The proposed research comprises applications of physics (plasmonics, optics, fluid dynamics), biotechnology (molecular design and interaction), bioinformatics (sequence alignment) and engineering (instrument design and validation). To achieve the ultimate goal of the project within the 36 months duration, the project will be implemented in a step-by-step approach, with gradual increases in complexity. Each of these single steps represent a very valuable result that will lead to important contributions to the different research fields and industrial applications. In particular, PROSEQO is expected to demonstrate a new and disruptive technology for single-molecule DNA/RNA next generation sequencing (NGS) by applying the technology developments to the less complex aim of discriminating nucleic acids (i.e. only 5 different molecules rather than 20).

Targeted breakthrough, long term vision and objectives

Targeted scientific breakthrough: the 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.

Specific main objectives

Objective 1

Targeted scientific breakthrough: the 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
Objective 2

Targeted scientific breakthrough: the 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
Objective 3

Targeted scientific breakthrough: the 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

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