HAPIscreen - proof of concept. (A) Scheme of the assay setup using a digoxigenin-tagged aptamer (R06) and a biotinylated target RNA hairpin (TAR). The association of the two components is detected by using both Donor streptavidin (D) and Acceptor anti-digoxigenin (A) coated AlphaScreen® beads. The production of singlet oxygen upon laser excitation by D-phtalocyanin is monitored by the fluorescence emission of A-rubrene beads. (B) Results obtained when increasing concentrations of dig-R06 were added to A and D beads for different biot-TAR concentrations (from 0 to 40 nM as indicated on the right). (C) Secondary structures and/or sequences of the top part of the trans-activating responsive (TAR) RNA element of HIV-1 (biot-TAR), 5' end-extended TAR (rA-TAR), RNA aptamer R06 (dig-R06), domain II of the HCV Internal Ribosome Entry Site (biot-DII), primer anchor (dig-primer). The latter was synthesized with 2'-O-methyl residues except at two positions (underlined) where Locked Nucleic Acid residues were introduced in order to promote hybridization and to increase complex stability. Oligod(T3CT21) anchor was used for capturing rA-TAR or the candidates from the M1 or M2 SELEX. The former were captured with biot-dT and the latter with dig-dT oligonucleotides, respectively.
Dausse et al. Journal of Nanobiotechnology 2011 9:25 doi:10.1186/1477-3155-9-25