Label-free Biosensing by selective gating of fluorescent molecules through a membrane-spanning synthetic DNA origami nanopore by researchers in Denmark

A scientific collaboration led by researchers at iNANO/Aarhus University and Copenhagen University has resulted in the construction of a synthetic DNA nanopore capable of selectively translocating protein-size macromolecules across lipid bilayers. These discoveries can pave the way for applications involving the programmable cell perforation and potential size selective delivery of drugs, DNA or even proteins as well as label free biosensing.

In 2015 the first commercial nanopore DNA sequencing device was introduced by Oxford Nanopore Technologies Based on a synthetically engineered transmembrane protein, nanopore sequencing allows long DNA strands to be channelled through the central lumen of the pore where changes in the ionic current work as a sensor of the individual bases in the DNA. This technique was a key milestone for DNA sequencing and the achievement was only made possible after decades of research.
Since then, researchers have tried to extend this principle to build larger pores to accommodate proteins for sensing purposes, but a major challenge in this feat has been our lack of understanding how to redesign proteins pores into new functional machines. The researchers here used, a new technique utilizing artificial folding of DNA into complex structures, the so called 3D-origami technique that was first employed by the group of Jørgen Kjems in iNano in Aarhus University in 2009. It has demonstrated an unprecedented design space of nanostructures to mimic and extend biological assemblies.
In the newly published article in Nature Communication the researches now reports the creation of a large synthetic nanopore made from DNA, which is capable of translocating large protein-sized macromolecules across lipid bilayer separated compartments. In addition, a functional gating system were introduced inside the pore to enable biosensing of very few molecules in solution. Lastly the pore was equipped with a set of controllable flaps allowing programmable and targeted insertion into membranes displaying particular signal molecules. The nanopore was extensively characterised by state of the art techniques including cryo EM.

Using EM allowed us to even see directly the nanoscale motions of the flaps says Rasmus Thomsen from Kjems group in Aarhus who did most of the measurements.

With the use of powerful single molecule optical microscopes of Hatzakis Group at the Department of Chemistry & Nanoscience center it was possible to follow for the first time the flow of molecules through individual nanopores. To do this the two teams filled model cells (liposome membranes of nanometer dimensions) with small or large fluorescently labelled molecules and recorded the nanopore perforation and molecules efflux in real time. By introducing a controllable plug in the pore, it was possible to size-selectively control the flow protein-size molecules and, in a proof-of-principle experiment, demonstrate label-free, real time, bio-sensing of a trigger molecule.

Our state-of-the-art single molecule microscopy allowed us to directly observe for the first time not only the nanopore docking but also the flow of proteins through the pore and thereby characterize the DNA nanopore and the efficiency of the targeting says Mette Galsgaard Malle from Hatzakis Group at University of Copenhagen who did the single molecule studies and statistical analysis.

In the future, this mechanism will potentially enable insertion of the sensor specifically into diseased cells and allow diagnosing of single cell level.

More information

Funding:
The work was funded by the Villum foundation to the BIONEC centre, the Villum foundation Young Investigator fellowship the Carlsberg Foundation (Distinguished associate Professor) and the Danish National Research Foundation to the CellPat Centre.

Research was a large cross-institution effort, carried out by researchers from Interdisciplinary Nanoscience Center (iNANO) at Aarhus University, from Department of Chemistry & Nanoscience center of University of Copenhagen as well Department of Physics, Chemistry and Pharmacy of University of southern Denmark and the department of physics at the Technical University of Munich. Nikos Hatzakis group is member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen and associated with the NovoNordisk center for Protein research CPR.

Read more about the results published on 11th December 2019 in Nature Communications here.

Contact:
Professor Jørgen Kjems
Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics
Aarhus University
JK@mbg.au.dk  - +45 87155494 –

Associate Professor Nikos Hatzakis
Nano-Science Center and Department of Chemistry
Copenhagen University
Hatzakis@chem.ku.dk  - +45 50202951 –