DNA nano-adapters: stimulus for single-molecule DNA sequencing

Photo: Graphic showing the immobilization strategy

Immobilization strategy: DNA origami (grey rectangles) equipped with a fluorescent dye (red) occupy the small holes in the metal film (ZMWs) in a way that only one adapter fits per cavity; ©Braunschweig University of Technology

Scientists at the Braunschweig University of Technology have developed tiny adapters that allow the coupling of molecules to nanostructures and their precise positioning on the scale of a millionth of a millimeter. This development is of relevance especially for DNA sequencing, which is considered the key technology for the analysis of inherited diseases.

For DNA sequencing, individual nucleotides are analyzed which are the building blocks of DNA. “Monitoring the incorporation of single nucleotides into a full DNA strand in real-time is a revolutionary method”, Prof. Philip Tinnefeld explains. “It is almost a live broadcast”. Special proteins, the so-called DNA polymerases, incorporate the nucleotides in a zipper like fashion to build a double stranded DNA strand. In order to observe this process and extract the order of nucleotides, scientists employ special cover slides. A glass slide is coated with a thin metal film that contains tiny holes, so-called zeromode waveguides (ZMWs).

“The challenge for this application is to equip each of these nano-holes with exactly one polymerase that utilize the nucleotides”, Tinnefeld says. Usually, these biomolecules are deposited randomly in the ZMWs, which results in many empty ZMWs while others contain multiple polymerase molecules. Even for the optimal situation, only 37 % of the holes can be used, as the expert for Nano-Bio-Sciences explains.

His research group now achieved a more efficient usage of the ZMWs by developing a new binding strategy. For this, the nano-experts from the Institute for Physical and Theoretical Chemistry in the Laboratory of Emerging Nanometrology (Braunschweig University of Technology) could use their experience of working with the so-called DNA origami technique: the Braunschweig scientists literally fold precisely fitting structures from single viral DNA strands. The nano-adapters were designed such that exactly one DNA origami can bind in every ZMW. The nano-adapters additionally provide docking points for functional units, like fluorescent dyes or the polymerase molecules that are used for DNA sequencing. “With our novel strategy, we connect single molecules via DNA origami with the lithographically fabricated ZMWs. This procedure can improve the efficiency of DNA sequencing and also be beneficial for applications in other areas of research like molecular electronics”, Tinnefeld summarizes.

COMPAMED.de; Source: Braunschweig University of Technology