Characterisation of Antibody-Binding RNAs Selected from Structurally Constrained Libraries

Constrained RNA libraries of limited sequence complexity were constructed and used to select RNA molecules binding to the antigen binding site of an anti-ferritin antibody. The sequences required as primer-binding sites for the selection cycle were designed to form a predictable secondary structure, which greatly facilitated the characterisation of the secondary structures of the selected RNAs. RNA-antibody interactions were studied by real-time interaction analysis to study the dynamic aspects of binding and by circular dichroism spectroscopy to search for conformational changes upon binding. The selected RNAs were analysed with a binding site sequestering assay and were shown to compete with ferritin for binding to the antigen-binding site. The experiments described here indicate that the introduction of strong structural constraints does not have to interfere with the ability to select tightly and specifically binding RNA-molecules.