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RESEARCH PRODUCT

Rupture Force of Single Small Drug Molecule Binding a Split Aptamer

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Aptamers are specific oligonucleotides (DNA or RNA) which bind small inorganic or organic molecules, large proteins or cells. In particular, the high affinity of aptamers is expected to lead to a new class of therapeutic reagents. Thus the detection and characterization of binding strength of small molecules is important for drug and medical research. Atomic force spectroscopy (AFS) with a force resolution in the piconewton range is a valuable tool for studying interactions on a single molecular level. The detection of very small target molecules less than 500 Dalton is characterized by only a few hydrogen interactions between the aptamer and the target molecules. Thus tiny rupture forces well below 100 pN are predictable. For AFS the target molecules as well as the aptamer probes are typically immobilized on the AFM-tip or sample surface, respectively. This concept suffers from the need to chemically manipulate or label the target analyte by binding sites that interact with surfaces. We solved this problem by using a split aptamer probe. Both components of the aptamer were immobilized on the SFM-tip and sample surface, respectively. During the AFS experiment the split aptamers form defined binding pockets for the free analyte. The concept of using a split aptamer allowed the detection of the binding of Adenosine mono-phosphate (AMP) molecules being label-free. We found an increase in rupture force of 11 pN in the presence of AMP molecules in both binding pockets. The route to use a split aptamer probes in AFS enables us to determine precisely the dissociation constant of the AMP-aptamer system (3.7 ± 1.5 μM) by increasing the AMP concentration in solution. The concept of a split aptamer binding single small target also worked for the cocaine and antibiotics molecules.