Construction and Single-Molecule Characterization of DNA Hairpin Constructs Based on the TAR RNA Sequence



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Optical tweezers have been used previously to examine the folding and unfolding of single molecules, revealing the effects of force, ionic strength, proteins, and small molecules on the kinetic and thermodynamic parameters of biomolecule stability. Here, we examine DNA hairpin constructs related to the Trans-Activator Response element (TAR) RNA sequence of HIV. In an optical tweezers experiment, a bead at one end of a duplex DNA assembly is attached to a micropipette, while a bead at the other end is held in an optical trap, thus allowing investigators to relate picoNewton forces necessary to unfold the DNA with corresponding nanometer changes in length. The force-versus-extension data are used to elucidate DNA folding and unfolding processes. Our DNA construct design contains a central hairpin structure with long double-stranded "handles" attached to both the 3’ and 5’ end of the hairpin. The DNA “handles”, each approximately 3 kb in length, were synthesized by PCR using biotin- and digoxigenin-labeled primers that serve to attach the final construct to 5 μm-diameter beads for use with the optical tweezers. The smaller central DNA hairpin sequences were obtained via automated solid-phase organic synthesis. After purification of each piece and assembly with a linker oligonucleotide, the 5’ Handle and the hairpin structure were ligated together and purified by agarose gel electrophoresis. This product was then itself ligated to the 3’ Handle and again purified and characterized. Individual molecules of the final hairpin constructs, approximately 6 kb in length, were bound to streptavidin or anti-digoxigenin labeled beads, and their unfolding behavior was studied using optical tweezers. In all, we studied three different DNA constructs, each possessing varying degrees of complementarity between the two sides of the hairpin stem. Our initial results are reproducible and show that the greater number of complementary bases within the hairpin, the greater the stability and the higher the force needed to stretch the hairpin into a single strand conformation.



DNA Hairpins, Optical Tweezers, Single-molecule experiments, DNA duplex model, TAR RNA Hairpin