The hairpin ribozyme belongs to a group of small catalytic RNA structures which is derived from the (-)-strand of the Tobacco ring spot virus satellite RNA. Here, we propose an electrochemical detection system for monitoring of the ribozyme activity using electroche¬mical impedance spectroscopy (EIS). For this purpose, a thiolated RNA substrate is self-assembled at a gold surface. Incubation with the hairpin ribozyme leads to the formation of Watson-Crick base pairs between the ribozyme and its complementary substrate and thus to an assembly of the ribozyme at the RNA substrate monolayer. Cleavage of the substrate which is leading to a disintegration of the ribozyme/substrate complex can be initiated by addition of Mg2+ ions.
The cleavage reaction is monitored by EIS by studying the modulation of the charge transfer resistance of a free-diffusing redox mediator ([Fe(CN)6]3-/4-]. The access of the free-diffusing and negatively charged redox mediator is modulated due to the chemisorption of the thiolated RNA and the subsequent formation of the RNA/ribozyme complex, which manifests as an increase in the charge transfer resistance in the impedance signal. Upon addition of Mg2+ ions the charge transfer resistance decreases again due to the cleavage reaction.
This strategy can be applied for the selection of a specific ribozyme sequence from a RNA library. This concept is evaluated by immobilizing the RNA substrate at a macroscopic gold electrode surface, assembling of the complementary hairpin ribozyme structure at the monolayer and initiating the cleavage reaction by addition of Mg2+ ions. The products of the cleavage reaction, namely the ribozyme and the cleaved part of the RNA substrate, are collected. The RNA is then reverse transcribed into cDNA, amplified by means of PCR and analyzed by sequencing. First results are showing that the ribozyme which was previously assembled at the RNA substrate modified surface was found in the collected solution.
The authors are grateful to the European Commission for financial support in the framework of the project INTCHEM (MEST-CT-2005-020681). The authors are grateful to Dr. Bettina Appel for synthesis of the RNA substrate.