Angewandte Chemie International Edition
Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Angewandte Chemie International Edition ,
Searching for Traces
Detection of the tiniest quantities of proteins by means of aptamers and PCR
Sometimes a few molecules of a protein are enough to control important physiological processes in a cell or to trigger a disease. While the detection of tiny amounts of DNA has become a standard technique, thanks to the polymerase chain reaction (PCR), there has been no corresponding technique for detecting protein molecules with the same degree of sensitivity. Scientists at the University of Alberta in Canada have now developed an ultra-sensitive method for detecting proteins. It is based on short DNA probes (aptamers) that bind specifically to a desired protein and their multiplication through PCR.
In order to test their new technique, the researchers led by Chris Le selected HIV-1 Reverse Transcriptase (HIV-1 RTase) as their target protein. This enzyme plays an important role in the life cycle of the human immunodeficiency virus. To detect this protein, they used a specific aptamer that binds to it. Aptamers are three-dimensionally folded short chains of DNA building blocks that recognize and bind to other nucleic acids, protein molecules, or even small organic molecules with high specificity. Today, there are methods to generate such aptamers as needed. This works by searching for “hits” from a huge number of random DNA sequences, and amplifying the selected sequence. The researchers chose a specific aptamer of known sequence for their target protein, HIV-1 RTase. If this protein is present in a sample, the aptamer binds to it. The researchers then use capillary electrophoresis to separate the aptamer–RTase complexes from the other components in the sample and from unbound aptamer. This separation technique relies on the different speeds of different molecules traveling through an extremely fine tube along an electric field. The fraction containing the aptamer–RTase complexes is then subjected to PCR. Beginning with starter DNA that specifically recognizes the sequence of the aptamer, copies of the aptamer are made. The number of aptamer molecules is thus multiplied and they can then be detected. The researchers were thus able to detect the small number of only 180 molecules of RTase. This puts the detection limit of the aptamer method several orders of magnitude lower than conventional techniques.
Since aptamers can be generated to fit nearly all proteins, this technique is universally applicable for the detection of proteins. The use of calibration curves makes quantification possible.