Periodic Reporting for period 1 - Origami-SEQ (SINGLE-MOLECULE DNA SEQUENCING THROUGH DNA ORIGAMI NANOANTENNAS.)
Okres sprawozdawczy: 2017-04-01 do 2019-03-31
The objective of OrigamiSeq is to create an experimental platform that will allow scientists to study how individual biomolecules works. Among the most important techniques that allow us to study isolated biomolecules are single-molecule fluorescence techniques. One of the main challenges that faces the application of single molecule fluorescence techniques in biology is the concentration barrier. This refers to the big difference in concentration required to detect a single molecule and the concentration at which biomolecules work. The density of molecules demanded for biomolecules to work efficiently are usually at least 1000 times higher than that required to achieve single molecule detection. This means that if we use the biomolecules, or their substrate, at their working concentration, we will detect more than one molecule at a time, therefore losing the single molecule information we are seeking to obtain.
To solve this problem we plan to use DNA origami nanoantennas. It is known, due to a combination of physical and chemical-physical phenomena, that a fluorescent molecule placed in the vicinity of a metal can experience an enhancement of its fluorescent emission. On the other hand, the DNA origami technique permits to build nanometer size structures with virtually any kind of shape and functionality. Our group combine both technologies to build nanostructures that can take advantage of this effect to increase the fluorescence signal coming from a single fluorescent molecule. The heart of the so-called DNA origami nanoantenna are two metal nanoparticles brought in proximity, combined with the ability to place selectively molecules in the hotspot where the enhancement is the highest. Since the signal coming from the molecule in the hotspot is far more intense that the signal coming from molecules out of it, metallic nanoantennas allow the detection of a single molecules even when several molecules at present at a time. Using this idea, we will use DNA origami nanotechnology to construct nanoantennas and selectively immobilize a biomolecule in the hotspot to study its behavior at concentrations compatible with their working concentration. We plan to develop a single-molecule DNA sequence technique in order to probe the viability of our idea. The long-term objective is to provide to the scientific community with a general platform with a high versatility that can be used to study a huge variety of biomolecules.
In order to understand how they perform their work, it is of crucial importance to study the molecules that plays key roles in life at the single molecule level. Better understanding of their function will lead us to better understanding of key features of life but also on how their malfunction is related to disease. Therefore paving the way to develop better future treatments for diseases. Besides, the project includes the development of a single-molecule DNA sequencing technology, with potential applications in diagnosis.
To solve this problem we plan to use DNA origami nanoantennas. It is known, due to a combination of physical and chemical-physical phenomena, that a fluorescent molecule placed in the vicinity of a metal can experience an enhancement of its fluorescent emission. On the other hand, the DNA origami technique permits to build nanometer size structures with virtually any kind of shape and functionality. Our group combine both technologies to build nanostructures that can take advantage of this effect to increase the fluorescence signal coming from a single fluorescent molecule. The heart of the so-called DNA origami nanoantenna are two metal nanoparticles brought in proximity, combined with the ability to place selectively molecules in the hotspot where the enhancement is the highest. Since the signal coming from the molecule in the hotspot is far more intense that the signal coming from molecules out of it, metallic nanoantennas allow the detection of a single molecules even when several molecules at present at a time. Using this idea, we will use DNA origami nanotechnology to construct nanoantennas and selectively immobilize a biomolecule in the hotspot to study its behavior at concentrations compatible with their working concentration. We plan to develop a single-molecule DNA sequence technique in order to probe the viability of our idea. The long-term objective is to provide to the scientific community with a general platform with a high versatility that can be used to study a huge variety of biomolecules.
In order to understand how they perform their work, it is of crucial importance to study the molecules that plays key roles in life at the single molecule level. Better understanding of their function will lead us to better understanding of key features of life but also on how their malfunction is related to disease. Therefore paving the way to develop better future treatments for diseases. Besides, the project includes the development of a single-molecule DNA sequencing technology, with potential applications in diagnosis.
Our approach to develop a single-molecule sequencing technique based on DNA origami nanoanntenas relies on the ability to follow the activity of a DNA polymerase when it perform its activity. Therefore, one of the main challenges of the project is to obtain active DNA polymerases working on top of DNA origami structures. During the duration of the project, special effort was done in order to develop strategies to allow for immobilization of complex DNA polymerase complexes in DNA origami structures.
We successfully developed several approaches to immobilize a DNA polymerase on top of origami structures. Particularly challenging was to probe that the DNA polymerases immobilized on this structures were indeed active. In order to do so we developed a single-molecule activity assay that finally prove the feasibility of our approach.
In another vain, the other pillar of the project relies on the possibility of reach fluorescent enhancement in different colors. For this purpose, our group designed silver-base DNA origami nanoantennas able to reach fluorescent enhancement in the entire visible light spectrum.
No exploitable results and scientific publications were obtained during the duration of the project, but the results were presented in different scientific workshop and congress. Besides, news and scientific results were posted in a website (https://origamiseq.wordpress.com) and in a Facebook profile (Origami-Seq Msca).
We successfully developed several approaches to immobilize a DNA polymerase on top of origami structures. Particularly challenging was to probe that the DNA polymerases immobilized on this structures were indeed active. In order to do so we developed a single-molecule activity assay that finally prove the feasibility of our approach.
In another vain, the other pillar of the project relies on the possibility of reach fluorescent enhancement in different colors. For this purpose, our group designed silver-base DNA origami nanoantennas able to reach fluorescent enhancement in the entire visible light spectrum.
No exploitable results and scientific publications were obtained during the duration of the project, but the results were presented in different scientific workshop and congress. Besides, news and scientific results were posted in a website (https://origamiseq.wordpress.com) and in a Facebook profile (Origami-Seq Msca).
During the two years of duration of the fellowship most of the scientific objectives has been fulfilled. Protocols for immobilization of DNA polymerases in DNA origamis has been developed, which is itself a challenging task due to the unspecific binding of DNA polymerases to DNA. Besides, proof of concept of fluorescence intensity enhancement in different colors by DNA origami nanoantennas was shown, and we develop a new single-molecule assay to track activity of φ29 DNA polymerase. Unfortunately, the final aim of performing single-molecule real time DNA sequencing was not achieved, nevertheless, the objectives and milestones that will eventually lead to this aim were reached. Overall, our results pave the way to the utilization of DNA origami nanoantennas as single-molecule fluorescent platforms to develop bioassays.
We hope that the scientific milestones reached in this project will eventually lead to new technologies on key area as photonics. Although the final aim of generating a new single-molecule DNA sequencing technology was not reached, we are confident that the new created knowhow will help us to generate eventually this new single-molecule DNA sequencing technology. This technology has potential applications in diagnosis and health, thus targeting an Horizont 2020 Key priority for 2016-2017: Promoting healthy ageing and personalized healthcare. The European competitiveness would also be potentiated in this case, since this technology can be licensed to European companies, giving a competitive edge that can secure growth and jobs in the EU and maintaining the European status quo of high-level-knowledge countries
We hope that the scientific milestones reached in this project will eventually lead to new technologies on key area as photonics. Although the final aim of generating a new single-molecule DNA sequencing technology was not reached, we are confident that the new created knowhow will help us to generate eventually this new single-molecule DNA sequencing technology. This technology has potential applications in diagnosis and health, thus targeting an Horizont 2020 Key priority for 2016-2017: Promoting healthy ageing and personalized healthcare. The European competitiveness would also be potentiated in this case, since this technology can be licensed to European companies, giving a competitive edge that can secure growth and jobs in the EU and maintaining the European status quo of high-level-knowledge countries