Title

DESIGN AND SYNTHESIS OF A BISTABLE APTAMER BASED THREE-SEGMENT SWITCH AS A DIAGNOSTIC INDICATOR FOR HIV-1 NCp7 DETECTION

Date of Award

May 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Philip N. Borer

Second Advisor

Mark S. Braiman

Keywords

Aptamer, Biosensor, DNA, HIV-1, Ncp7, three segment

Subject Categories

Physical Sciences and Mathematics

Abstract

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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