DESIGN AND SYNTHESIS OF A BISTABLE APTAMER BASED THREE-SEGMENT SWITCH AS A DIAGNOSTIC INDICATOR FOR HIV-1 NCp7 DETECTION
Date of Award
Doctor of Philosophy (PhD)
Philip N. Borer
Mark S. Braiman
Aptamer, Biosensor, DNA, HIV-1, Ncp7, three segment
Physical Sciences and Mathematics
The HIV-1 nucleocapsid protein (NCp7) is an attractive anti-retroviral drug target. A unimolecular oligonucleotide sensor is comprised of three segments of DNA joined at a branch. NC is recognized by a DNA aptamer incorporated in the Probe (P) segment of the sensor. This aptamer forms a 1:1 complex with NCp7 and Kd = 17nM (0.2M NaCl, 0.05M phosphate buffer, pH 7.0); an affinity 125 times higher than any of the other sensor segments. In the target-free state, the aptamer is concealed by base-pairing with the complementary Cover (C) segment. The third segment (Toggle, T) is an imperfect mimic of P lacking the NC-recognition element; T competes with P to base-pair with C in a rapid two-state equilibrium between different conformations. A fluorophore at the P-5’-terminus and quencher at the C-3’-terminus are used to estimate the populations of the two conformations of this nanoscale switch. A key issue is to design a switch that favors the P:C duplex, with an equilibrium constant K1 = [T:C]/[P:C] = 0.01 to 0.1 by optimizing the sequences of C and T. This switch has minimal fluorescence in the absence of target. Upon adding target, the equilibrium shifts toward the T:C form, triggering a large increase in fluorescence. Moving the fluorophore from the P- to the T-terminus creates a turn-on switch suitable for high-throughput screening (HTS) of anti-AIDS drug candidates in simple mix-and-measure assays. This dissertation details the procedure for preparing diagnostic NC-switches in 90%+ purity, and evidence that NC induces the transition between the stable states of the switch within seconds. Similar switches thermodynamically tuned for specific targets in diagnostics and drug discovery should be useful for a broad spectrum of chemical and biological agents.
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Iyer, Raghuvaran Mani, "DESIGN AND SYNTHESIS OF A BISTABLE APTAMER BASED THREE-SEGMENT SWITCH AS A DIAGNOSTIC INDICATOR FOR HIV-1 NCp7 DETECTION" (2020). Dissertations - ALL. 1223.