In this month’s Nature Biotechnology, German and American scientists led by Michael Famulok, University of Bonn, report that they have engineered catalytic RNAs that can measure protein-protein interactions in real time. These chimeric ribozymes may prove to be extremely useful in high-throughput screens for small-molecule drugs.
First author Joerg Hartig et al. based the chimeras on the self-cleaving hammerhead and hairpin varieties of catalytic RNA. Into these they incorporated a fluorescent reporter and a fluorescence quencher on either side of the cleavage site. This allows catalytic activity to be measured as an increase in fluorescence as the fluorophore is released from the quencher. Into this ribozyme the authors then introduced RNA sequences that are complementary to DNA aptamers-small regions of nucleic acids that recognize specific molecular structures, much like antibodies. In this case the authors inserted a sequence that complements a human a-thrombin aptamer. In this configuration, the ribozyme is poisoned by the aptamer and activity can only be restored by removing the DNA from solution or-and this is key to their technique-by adding sufficient “antigen,” in this case thrombin, to mop up the offending aptamer. In testing this ribozyme configuration with 13 different proteins; a-thrombin yielded substantial fluorescence while its close relative g-thrombin failed to activate catalysis.
But what of protein-protein interactions? Well, removing the a-thrombin either physically or by adding another protein that binds to it, should, in theory, release the aptamer, allowing it to bind and inhibit the ribozyme. In fact, this is exactly what happens. The authors titrated the ribozyme/aptamer/thrombin mixture with the thrombin inhibitor hirudin, and each successive addition quenched the fluorescence more.
This is not the first paper to tout the use of aptamers for detection systems (see related news item ). As Larry Gold of SomaLogic Inc. in Boulder, Colorado, points out in an accompanying News and Views, this method shows great potential but is currently limited by a rather small pool of available aptamers. Hopefully, this will soon change.—Tom Fagan
- Hartig JS, Najafi-Shoushtari SH, Grüne I, Yan A, Ellington AD, Famulok M. Protein-dependent ribozymes report molecular interactions in real time. Nat Biotechnol. 2002 Jul;20(7):717-22. PubMed.
- Gold L. RNA as the catalyst for drug screening. Nat Biotechnol. 2002 Jul;20(7):671-2. PubMed.