An oligonucleotide molecular switch sensor is designed to target the HIV-1 nucleocapsid protein that is involved in recognition and packaging of genomic RNA during viral replication. The switch has three segments of DNA that join at a branch (see Figure). The recognition element is a DNA aptamer (Probe) segment. Results show that the affinity of the aptamer for NCp7 is 125 times higher than any of the other switch segments. In the OFF state (Figure), the aptamer is inactive due to base-pairing with the complementary (Cover). The third segment (Toggle) is similar to Probe but lacks the recognition element; thus Toggle and Probe compete to base-pair with Cover, setting up a two-state ON/OFF equilibrium. Distinction between the two conformations is achieved by covalent labeling with a fluorophore-quencher pair (Figure). A key design parameter for the switch is an equilibrium constant K1 = [ON]/[OFF] = 0.01 to 0.1 set by altering the sequences of Cover and Toggle to slightly favor the binding of Probe by Cover in the absence of target. Addition of a small concentration of the target pulls the equilibrium toward the ON form causing an increase in fluorescence up to 90-95% of the saturation value. Such reversible and rapidly equilibrating sensors show promise as real-time diagnostic indicators for detection of toxins and mix-and-measure assays for high-throughput screening (HTS) of drug candidates for protein targets.

Aptamer based DNA sensor for HIV-1 NCp7

248th National Meeting (2014)

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