Development and Characterization of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery

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A unimolecular bistable oligonucleotide, a branched switch, consisting of three unique segments is designed based on an aptamer that selectively and specifically binds to HIV-1 nucleocapsid protein (NCp7). This novel DNA sensor comprises cover, toggle and probe sequences that can form individual hairpins. In the absence of any base pairing, the molecule resembles a Y-shaped structure with the three different strands joined at the fork by 5-Me dC brancher. The probe sequence is an aptamer that binds NCp7 protein with a high affinity (Kd= 15nM). The novel DNA sensor is thermodynamically stable in two conformations, ON and OFF, where covalent labels, a Cy3 at the 5’-end of toggle and a Dabcyl at the 3’-end of cover, allow readout of the conformational equilibrium. The ON form has the highest fluorescence intensity and dominates the sensor equilibrium in the absence of NCp7. As the concentration of NCp7 increases, fluorescence decreases due to contact quenching of Cy3 by Dabcyl. The sensor reverts to the ON form when a potential drug competitor binds to the aptamer binding site of NCp7, thereby releasing the switch. Due to uncertainties in the calculations, especially the free energy contribution from dyes interacting at the termini of base-paired strands, the predicted equilibrium constant for a candidate switch (K1) must be experimentally tested. In this project, various switch molecules with different equilibrium constants (K1=0.001-0.1) were designed using Visual-OMP and then synthesized using a DNA synthesizer and an enzymatic ligation method. The switch purity is above 90% based on ESI-MS analysis. The quenching of candidate switches with and without NCp7 can be analyzed using a simple plate reader. Switches with a high contrast ratio will be used to screen anti-HIV drug candidates.