Designer proteins fold DNA

Biophysicists construct complex hybrid structures using DNA and proteins – opportunities for research in cell biology

Double-stranded DNA, by Ella Maru Studio and Dietz Lab / TUM

Florian Praetorius and Hendrik Dietz of the Technical University of Munich (TUM) have developed a new method that can be used to construct custom hybrid structures using DNA and proteins.

The method opens new opportunities for fundamental research in cell biology and for applications in biotechnology and medicine.

DNA is an excellent building material for nanostructures. Folding DNA to create three-dimensional shapes ("DNA origami") is long-established but has limits, explains Dietz.

To start with, the "construction work" always takes place outside of biological systems and many components must be chemically synthesised.

"Creating user-defined structures inside a cell, in sizes of 10–100 nanometers, remains a great challenge," Dietz said.

The team's newly developed technique allows the researchers to use proteins to fold double-stranded DNA into the desired three-dimensional shapes.

Then both the DNA and the required proteins can be genetically encoded and produced inside cells.

Proteins act as staples

Designed "staple proteins" based on TAL effectors are the key to the method. TAL effectors are produced in nature by certain bacteria that infect plants and are able to bind to specific sequences in the plant DNA, thereby neutralising the plant's defence mechanisms.

"We've constructed variants of the TAL proteins which simultaneously recognise two custom target sequences at different sites in the DNA and then basically staple them together," said Dietz. "This was exactly the property we needed – proteins that can staple DNA together."

The second component of the system is a DNA double strand containing multiple binding sequences that can be recognised and linked by a set of different staple proteins. "In the simplest case, a loop can be created by binding two points to one another," Praetorius explains.

"When several of these binding sites exist in the DNA, it's possible to build more complex shapes."

An essential aspect of the researchers' work was determining a set of rules for arranging the staple proteins and how to distribute the binding sequences on the DNA double strand to create the desired form.

New tools for fundamental research

Staple proteins serve as anchor points for additional proteins. A method referred to as genetic fusion can be used to attach any functional protein domain desired.

The hybrid structures made of DNA and proteins then function as a three-dimensional framework which can put the other protein domains into a particular spatial position.

All the building blocks for the DNA protein hybrid structures can be produced by the cell itself and then assemble themselves autonomously.

The researchers were able to produce the hybrids in environments resembling cells starting from genetic information. "There is a fairly high probability that this will also work in actual cells," said Dietz.

The new method paves the way for controlling the spatial arrangement of molecules in living systems, which allows probing fundamental processes.

This was exactly the property we needed – proteins that can staple DNA together.

For example, it's assumed that the spatial arrangement of the genome has a substantial influence on which genes can be read and how efficient the reading process is. The intentional creation of loops using TAL-DNA hybrid structures in genomic DNA may provide a tool for investigating such processes.

It would also be possible to geometrically position a series of proteins inside and outside the cell in custom ways to investigate the influence of spatial proximity, for example on information processing in the cell.

The spatial proximity of certain enzymes could also make processes in biotechnology more efficient.

Lastly, it is conceivable to utilise protein-DNA hybrid structures for example to better stimulate the immune response of cells, which can depend on the precise geometrical arrangement of multiple antigens.

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