Impedimetric Detection of Hairpin Ribozyme Activity

Abstract

Electrochemical impedance spectroscopy is successfully utilized for label-free monitoring of the cleavage reaction of a RNA substrate by a complementary hairpin ribozyme. The formation of the RNA substrate/ribozyme complex is increasing the negative charge at the interface and is modulating the kinetic of the redox conversion of a negatively charged redox mediator (e.g. [Fe(CN) 6 ] 3À/4À ) hence leading to an increase in the charge transfer resistance. Upon addition of bivalent cations such as Mg 2 + ions the conformation of the ribozyme is changed and the catalytic cleavage of the RNA is monitored by a decrease of the charge transfer resistance.

Preparation of modified long-mer RNAs and analysis of FMN binding to theypaAaptamer fromB. subtilis

“…Thus, we have started setting up an electrochemical assay to follow binding and re-binding of the aptamer in response to the FMN oxidation state. 10,48 Furthermore, we intend to use Terahertz spectroscopy to observe local changes in the water network of the aptamer upon folding, as well as EPR and/or NMR spectroscopy with nitroxide spin labels or fluorine nuclei as folding reporters to follow structural rearrangements of the aptamer in response to FMN.…”