Synthesis and Single-Molecule Characterization of a DNA Hairpin Construct Based on the TAR RNA Sequence

Abstract

Optical tweezers are used 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 bimolecular stability. Here, we examine a DNA hairpin construct related to the trans-activation responsive region (TAR) RNA sequence of HIV-1. 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. Force extension/relaxation 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 hairpin sequence was obtained via solid-phase organic synthesis. After purification 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 ligated to the 3’ handle and again purified. Individual molecules of the final hairpin construct, approximately 6 kb in length, were bound to streptavidin or anti-digoxigenin-labeled beads and their unfolding behavior was studied using optical tweezers.