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H2020

HYBRIPORE Report Summary

Project ID: 655660
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - HYBRIPORE (Hybrid DNA-protein nanopores with large and uniform pore sizes)

Reporting period: 2015-07-01 to 2017-06-30

Summary of the context and overall objectives of the project

Every living cell transports thousands of molecules per second across its membrane and into different compartments. Proteins actively and selectively regulate this transport in the majority of cases and they are vital to cell functioning. Over the past years some of the pore-forming proteins involved in transport have been isolated or expressed in vitro to develop biosensors for heavy metals and neurotransmitters, and DNA sequencing technologies with a high selectivity and extreme sensitivity.

An important bottleneck in technologies based on these protein nanopores is the inflexibility of the self-assembly of the pore-forming proteins. As a direct consequence, it is currently impossible to reliably make smaller or larger pores than the size that is dictated by the protein itself: a heptameric protein like haemolysin will always assemble into a heptameric pore. A second consequence is that the relative order of these seven subunits cannot be imposed. This limits the development of novel sensors and artificial enzymes that require more than one altered subunit.

To overcome these limitations our key objective is to design a molecular scaffold that can template both the number and the relative order of protein subunits that later form a nanopore (Fig. 1). Our scaffold is made of DNA and can be constructed accurately and reproducibly in a single step. It can be modified with proteins or parts of proteins that should be brought together in a nanopore, and it can be detached if required after the correct nanopore has formed.

Nanopore scaffolds will not only allow us to construct more advanced biosensors and DNA sequencing tools, but we can also use them to learn more about pore-forming proteins, about how they work and why they are vital to cells. We can use them to understand what pore sizes are preferred by various proteins and why, and we could discover which parts of a protein are essential for it to make a pore. Ultimately, these insights can help improve existing antibacterial therapeutics that target transport proteins and stimulate the discovery of new antibiotics.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have designed and constructed a molecular scaffold made of DNA that can template both the number and relative order of protein subunits.

Our design consists of twelve short single-stranded pieces of DNA that come together into a small ring with twelve arms onto which proteins can be attached (Fig. 1). This design has two key advantages over alternative designs. It is more flexible than the larger DNA origami structures based on helix bundles making it much easier to bring together two proteins attached at different binding sites without the need for long and floppy linker molecules. In addition, it does not require an excess amount of the short, protein-modified DNA molecules, of which the majority is incorporated into the scaffold and which may have adverse effects in applications. We used simulations to verify that our designed scaffold is sufficiently flexible to bring all arms together. In experiments, we have shown that the scaffold can be formed with more than 85% overall yield (based on all DNA molecules), that it is indeed a circle with twelve parts, as designed, and that it has the correct overall mass. Moreover, we have shown that our design can easily be extended to 14, 16 or 18 functional sites.

In the second phase of this project we attached various pore-forming proteins and peptides to our DNA scaffold. In the approach we have taken here (Fig. 2), all building blocks are first modified separately with the selected peptide through either a permanent or a cleavable linker, and then purified and analysed. This ensures that we can actually scaffolds large numbers of proteins or peptides despite the moderate yield of the reaction between DNA and protein. We have shown that our purified peptide-modified DNA can scaffold any number of peptides up to a maximum of twelve (for the smallest scaffold we made) with a high yield.

We then studied the nanopores that were formed by the templated peptides and the effect of the DNA scaffold on these pores. Using electrical recording of a planar lipid bilayer, in which a single nanopore can be probed at a time, we established that our DNA scaffold can stabilise peptide nanopores that are otherwise unstable. Moreover, we have shown that the scaffold can be used as a docking site for a variety of molecules, enabling repeated detection or sequencing of an analyte at ultralow concentration, and the in situ assembly of protein complexes near a nanopore sensor. When we built scaffolds containing more peptides than required in the pores that occur in nature, we could not observe larger nanopores. Instead, the peptides usually formed the same pores as they would in their natural protein environment. Apparently, the peptides we have tested so far are not sufficiently accommodating to adapt to the template offered by our scaffold. Hopefully, alternative peptides that we plan to investigate in the future will prove more able to assemble into differently sized pores.

Our results have been disseminated within the scientific community on various occasions: several talks at seminars, posters at conference dedicated to Biointerface Science and a second conference on Physics of Life, and we are preparing a paper about our scaffolded nanopores.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The possibility to template a peptide nanopore with a versatile scaffold that is nearly invisible in electrical recordings is a clear advancement beyond the current state of the art in protein nanopore technologies. Recently, researchers have shown that peptide nanopores can be scaffolded onto rigid scaffolds of a single size, but these peptides nanopores were already stable without a scaffold. Here, we have shown that DNA scaffolds can keep peptides nanopores stable against collapse and that they can be used to reversibly bind analytes or proteins in the vicinity of a nanopore. These results of a fundamental study into nanopores and DNA nanotechnology are not expected to be implemented into applications on a very short time, but they may have a strong impact on follow-up studies aimed at developing new advanced biosensors, and other fundamental studies into the structure and role of protein nanopores in various living organisms.

When constructed with the right peptides, scaffolded nanopores may be used to map out the natural conformations (A, B, Z) of pieces of double-stranded DNA under varying conditions, to sequence dsDNA in real time, and to detect epigenetic modifications, such as methylations, and other mutations. Improved, low-cost sequencing and mutation-detection techniques for dsDNA will have an enormous impact on diagnostics and forensics. In other, fundamental studies, the use of scaffolds for peptides and proteins will offer a unique tool to study protein assembly in solution and in membranes in a controlled way. This may lead to a better understanding of a wide range of oligomeric proteins, transport proteins and proteins that function in large multicomponent assemblies. These insights will have a big impact on our understanding of many diseases and thereby stimulate the development of new therapeutics.

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