Universal detector made of DNA building blocks
with pockets for two AMP molecules.
They then fix one half of the split aptamer to the tip of an atomic force microscope and the other to a support. When they then lower the tip and the halves come into contact, hydrogen bridge bonds form between individual bases of the two aptamer halves. If the tip is withdrawn, the joined aptamer is stretched like a spring. The force this produces can be measured: it increases with the strain until the halves tear apart at a certain force. In a second trial, before ripping apart, the researchers added a solution of the biomolecule adenosine monophosphate (AMP) to the system. In this way, two AMP molecules are placed in the free pockets; both then form hydrogen bridge bonds with the two halves of the aptamer. As a result of this bridge function, the AMP molecules reinforce the coherence between the two parts and therefore only tear apart at a much greater force. This difference enables the AMP to be detected.
To determine the rupture forces, the researchers repeated the measurements 1,000 times and determined a statistical mean which was around 39 piconewtons for the AMP-loaded aptamer, around 12 piconewtons higher than without the AMP. As a control, they used a mutated aptamer with a differently formed binding pocket in which the rupture force did not change. The binding strength between the AMP and the aptamer can also be readily determined by splitting the aptamer into two. To do this, the concentration of the AMP molecules in the solution was increased stepwise until approximately half of all tensile tests showed an increase in the tear force. The greater the concentration necessary for this, the smaller the binding strength is.
The new method is suitable not only for detecting certain molecules in a solution, but also for researching individual molecules, says Rüdiger Berger.
“For example, with a defined force you can pull the aptamer without tearing it and examine how the properties of the molecule-aptamer bond changes”, he continues. The target molecule could also be changed so that it forms, for example, only two hydrogen bridge bonds with the pocket instead of three. “This makes is possible to understand which bonds between the target molecule and the aptamer are significant”, he explains.
Knowledge of aptamers and their binding properties has great application potential. DNA fragments are already being used today for environmental analysis and in medical diagnostics; their uses as molecular tools and building blocks can be expanded even further with the new method thanks to the fresh insight, says Rüdiger Berger.
—Read more in Thi-Huong Nguyen et al., “Measuring Single Small Molecule Binding via Rupture Forces of a Split Aptamer,” Journal of the American Chemical Society 133, no. 7 (2 February 2011): 2025-27 (DOI: 10.1021/ja1092002)
