At the Max Planck Institute for Polymer Research in Mainz, Germany scientists are using aptamers and an atomic force microscope to detect the presence of individual molecules of specific substances. When aptamers bind to target molecules, the force needed to break them changes which then can be detected by the microscope. The new methodology allows for detection of a wide range of chemical compounds and offers a new option for studying molecular interactions.
Among the appropriate aptamers, the researchers in Mainz are looking for one that can be split into two parts in such a way that the target molecule bound in the pocket forms a bridge between the two halves. An aptamer such as this could be found mostly in pre-selection, explains Mark Helm of the Institute of Pharmacy at the Johannes Gutenberg University Mainz, co-author of the study. For their first trials with the universal detector, the researchers selected adenosine monophosphate (AMP) as the target molecule and an aptamer 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 If the tip is withdrawn, the joined aptamer is stretched like a spring.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 1000 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.
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Abstract in Journal of The American Chemical Society: Measuring Single Small Molecule Binding via Rupture Forces of a Split Aptamer