Final Report Summary - AMNA (Addressable Molecular Node Assembly - a Generic Platform of Nano-scale Functionalised Surfaces Based on a Digitally Addressable Molecular Grid)
The goal of our program has been to create a nano-technology platform based on a 100 nm size grid of addressable molecular building blocks ('nodes') to constitute a bottom-up modular approach to place functional groups at defined positions in space with sub-nm precision. In this way a bridge may be established between the traditional larger-scale nanotechnology based on lithography and the sub-nanometer molecular nanotechnology. Such a bridge is anticipated to provide enormous potential to future nano-technology as it could dramatically scale down the size of integrated units (integrated circuits) and other functionalities.
Important goals successfully achieved during the project:
A novel branching phosphoramidite monomer has been developed, and functionalised oligonucleotides, containing it as node, been synthesised. Systematic studies of oligonucleotide hybridisation have provided a platform for how supramolecular double-stranded nets of oligonucleotides may be constructed.
Systematic insertions of fluorescence markers and their energy transfer rates show how energy transfer may be used to gauge two-dimensional node structure. In order to optimise thermodynamic and kinetic hybridisation to error-free networks, libraries of orthogonal nucleobases sequences have been developed.
Systematic studies of oligonucleotide hybridisation have provided a platform for how supramolecular double-stranded nets of oligonucleotides may be constructed. This has been studied mainly with FRET, fluorescence and UV melting experiments and gel electrophoresis. Spreading / mixing / hybridisation behaviour of lipid-anchored DNA oligomers in lipid layer has been possible to follow with fluorescence microscope techniques.
A hexagon made up of linear as well as three-way oligonucleotides has been attached to a lipid bilayer. First successful DNA hexagon assembly with standard as well as novel trigonal DNA oligonucleotides has been proved and published. Novel analytical techniques for characterisation of this kind of DNA constructs developed and presented.
Systematic studies of oligonucleotide hybridisation have provided a platform for how supramolecular double-stranded nets of oligonucleotides may be constructed and studied mechanistically, mainly using FRET, fluorescence and UV melting experiments in combination with gel electrophoresis. The successful assembly and study of the first DNA bihexagon (DNA-'naphthalene') consisting of 10 trigonal oligonucleotides. In the article we used gel electrophoresis and but also AFM to assess the structure.
The same article also reports proof of principle that any edge in a system of a large number of DNA sequences may be uniquely addressed. DNA triplex recognition was used to demonstrate (using FRET probes) the addressability. The successful use of FRET also shows that we have distances short enough so that we will be able to use our DNA networks to transfer energy in predetermined paths.
We have published a fluorescence microscope study of lipid-anchored DNA oligomers in a lipid layer. Here we show how we can control spreading, mixing and release-behaviour of the lipid-modified oligonucleotides. This is important for future controlled mixing / hybridisation of oligonucleotides at lipid interfaces. We have completed a study in which lipid-modified DNA oligomers interact with lipid membranes. We show proof of insertion of lipid-modified DNA oligomers into membranes of different lipid compositions. Furthermore, we show that we can hybridise a complementary DNA to the membrane-anchored lipid-modified oligonucleotide. We also present structural information on the oligonucleotides at the lipid-water interface.
Further development of chemical ligation methods to produce covalently linked ring structures from the non-covalently linked hexagons, allowing structure verification by MS techniques, AFM etc. These new cross-linked hexagons may in the future serve as the nano-construct building-blocks rather than adding each oligonucleotide every time a network is constructed (i.e. pre-assembly of different hexagons that are then cross-linked, stored and subsequently used when needed).
Important goals successfully achieved during the project:
A novel branching phosphoramidite monomer has been developed, and functionalised oligonucleotides, containing it as node, been synthesised. Systematic studies of oligonucleotide hybridisation have provided a platform for how supramolecular double-stranded nets of oligonucleotides may be constructed.
Systematic insertions of fluorescence markers and their energy transfer rates show how energy transfer may be used to gauge two-dimensional node structure. In order to optimise thermodynamic and kinetic hybridisation to error-free networks, libraries of orthogonal nucleobases sequences have been developed.
Systematic studies of oligonucleotide hybridisation have provided a platform for how supramolecular double-stranded nets of oligonucleotides may be constructed. This has been studied mainly with FRET, fluorescence and UV melting experiments and gel electrophoresis. Spreading / mixing / hybridisation behaviour of lipid-anchored DNA oligomers in lipid layer has been possible to follow with fluorescence microscope techniques.
A hexagon made up of linear as well as three-way oligonucleotides has been attached to a lipid bilayer. First successful DNA hexagon assembly with standard as well as novel trigonal DNA oligonucleotides has been proved and published. Novel analytical techniques for characterisation of this kind of DNA constructs developed and presented.
Systematic studies of oligonucleotide hybridisation have provided a platform for how supramolecular double-stranded nets of oligonucleotides may be constructed and studied mechanistically, mainly using FRET, fluorescence and UV melting experiments in combination with gel electrophoresis. The successful assembly and study of the first DNA bihexagon (DNA-'naphthalene') consisting of 10 trigonal oligonucleotides. In the article we used gel electrophoresis and but also AFM to assess the structure.
The same article also reports proof of principle that any edge in a system of a large number of DNA sequences may be uniquely addressed. DNA triplex recognition was used to demonstrate (using FRET probes) the addressability. The successful use of FRET also shows that we have distances short enough so that we will be able to use our DNA networks to transfer energy in predetermined paths.
We have published a fluorescence microscope study of lipid-anchored DNA oligomers in a lipid layer. Here we show how we can control spreading, mixing and release-behaviour of the lipid-modified oligonucleotides. This is important for future controlled mixing / hybridisation of oligonucleotides at lipid interfaces. We have completed a study in which lipid-modified DNA oligomers interact with lipid membranes. We show proof of insertion of lipid-modified DNA oligomers into membranes of different lipid compositions. Furthermore, we show that we can hybridise a complementary DNA to the membrane-anchored lipid-modified oligonucleotide. We also present structural information on the oligonucleotides at the lipid-water interface.
Further development of chemical ligation methods to produce covalently linked ring structures from the non-covalently linked hexagons, allowing structure verification by MS techniques, AFM etc. These new cross-linked hexagons may in the future serve as the nano-construct building-blocks rather than adding each oligonucleotide every time a network is constructed (i.e. pre-assembly of different hexagons that are then cross-linked, stored and subsequently used when needed).
