Synthetic biology is a discipline with the bold aim to expand the scope of nature. It sees biology from the perspective of an engineer and asks how existing biomolecular structures, cellular processes or cells can be altered with the objective to achieve benefits in applications. These can range from biosensing over biomedicine to biocatalysis. An example is the engineering of a thermally stable enzyme that is used in a washing detergent. The higher aim is, however, not to replace small parts of an enzyme but to completely redesign cellular components or make them from scratch. This can be advantageous as it gives more control over the properties of the new synthetic products.

Our project falls within this category of synthetic biology. We will create synthetic versions of membrane proteins from the bottom up. Natural membrane proteins are important as they are involved in many aspects of life including the neural function, the perception of sensations, and the development of an immune response. Replicating some of the functions is however challenging with traditional building materials such as polypeptides. The issue is that the polypeptide chains do not always fold in the correct anticipated structures.

We will use DNA as a construction material to overcome the problem. DNA's natural role is to carry genetic information. It is composed of duplex of two strands held together by base pairs. But scientists have since discovered that the base pairing can be used to form non-natural structures.

Here we will produce a series of nanopores composed of DNA. The novelty is that the pores will achieve unprecedented control over transport across membranes. The new nanoscale pores will validate the concept that DNA is a suitable material. In addition, the pores will be exploited to build synthetic membrane compartments that can be used in cell biological research, or for the development of new anti-cancer drug systems.

Technical Abstract:
Pores are essential in biology as they mediate transport of matter across cell membranes. Given their highly precise functions, there is considerable potential to exploit membrane pores for biotechnology and biomedicine applications. But the naturally evolved protein structures with complex architectures can be difficult to engineer which limits their scope outside the native cellular context.

This project will pursue a radically different approach of building functionally advanced membrane pores with folded DNA nanostructures. DNA is one of the best materials for rationally designing nanostructures of defined geometrical architectures. Recently, we and others have overcome the hurdle of inserting a negatively charged DNA structure into a hydrophobic bilayer by equipping the nanopore with hydrophobic membrane anchors. Yet, the existing simple pore structure is of limited functional use.

Here, we will capitalize on the breakthrough and generate advanced pores for size and charge-selective transport that can be externally controlled. The pores will be of tunable size, charge properties, and activities, something not possible with any other bio-chemical building blocks.

The pores will be exploited in applications including the development of membrane compartments for light-triggered release of proteins cargo useful in cell biological research, and enzymatic membrane nanoreactors for the localized activation of anti-cancer pro-drugs.

The pores' construction and analysis will harness the ability of co-PI Prof. Hao Yan to create complex DNA nanostructures and the expertise of PI Dr. Stefan Howorka in engineering and characterizing DNA nanopores. The creation of synthetic membrane compartments can be used as precise light-controlled research tools in cell biology and later bioimaging and as powerful drug activation/delivery vehicles with selectively porous walls in cancer research, respectively.

Potential Impact:
The project will have an impact in the following three areas:

The first impact will be in basic science for cell biology to better understand signaling processes that are involved in the normal functioning of cells as well as in diseased tissues. Signaling cascades involve membrane surface receptors and intracellular kinases. The receptors' lateral position and number play an important role in cell activation. The new light-triggered research tools will give the required experimental control by determining when, where and how much active protein is being present. The new route can overcome limitations of other approaches for light control such as photo-caging or optogenetics that either require potentially harmful chemical modification of proteins or are not applicable to all proteins, respectively.

In a first stage, the new photo-release approach can be applied to target membrane proteins by releasing cognate ligands from vesicles. The light-controlled approach will improve the lateral and temporal resolution of release, and the control over the amount of protein compared to the current micropipetting approach used to eject protein solutions. It will also be less invasive. In a second stage, the new approach can release other, smaller biomolecules that do not have any chemical groups to attach photocages. The molecules include important neurotransmitters. For this application, the DNA pores will be designed to completely seal off- in the closed state- small compound transport and thereby overcome the low 5-10 fold difference in transport rates between the open and closed state of existing light-gated protein pores, as well as leakage in the off-state. When used in the context of neuronal cells, the DNA pore approach will yield light-controlled pre-synaptic vesicles with very high open-close differences for release, which is relevant in research. With further development, the light-gated approach may also be used to release protein inside cells which will be of interest for kinases. This will involve the take-up of vesicles by the endosome pathway and the engineering of the DNA pores to puncture the outer endosome membrane, something which is within reach. We expect that the research on signaling cascades will have an impact on drug discovery.

The second impact will be in targeted drug delivery and cancer therapy, and ultimately on improving the heath of people. Membrane vesicles that localize drugs to diseased tissues are FDA approved such as for anti-cancer drug doxorubicin. Encapsulating enzymes that activate pro-drugs represents the next-generation approach. It benefits from the amplification effect of enzymes that undergo millions of catalytic cycles and thereby drastically enhance the local concentration of drugs. Hence, we expect that the project will lead to further development of the synthetic vesicles for drug activation such as by equipping them with membrane-tethered antibodies for cell recognition and by testing them in vivo.

The third impact of the proposal is that it will help strengthen the industrial base by helping develop new research tools and drug delivery/activation agents in collaboration with companies.