Single Molecule Unfolding of RNA Hairpins

The TAR (trans activation response) element controls the life cycle of the HIV-1 virus as it initiates reverse transcription. Before proceeding with reverse transcription, the folded TAR hairpin must unfold to facilitate strand transfer. Chaperone assistance from the nucleocapsid (NC) protein can increase this rate-limiting step by 3000-fold. Interestingly, the TAR hairpin is not perfectly matched. It contains three loop-like “bubbles” and two non-canonical GU wobble pairs that destabilize the hairpin thermodynamically. We use optical tweezers to measure the force needed to unzip TAR variants with and without NC. To manipulate the hairpins for pulling with optical tweezers, the RNA hairpin is flanked by two long double-stranded DNA handles. One handle binds to a bead in the laser trap, while the other handle binds to a bead held by a micropipette, allowing us to pull on the construct and exert force on the hairpin. Handles were prepared by amplification of a generic plasmid by PCR with labeled primers; the RNA hairpin was transcribed from a DNA template by T4 RNA polymerase. After ligation of the pieces, gel purification allowed us to confirm the product and isolate the hairpin construct. Preliminary DNA pulling data obtained by optical tweezers confirm that the fully matched TAR hairpin is more stable than the canonical TAR, and that less force is required to pull apart a TAR hairpin with NC than one without. Strategic modifications to the sequence of the RNA hairpin allow us to examine the role specific structures and sequences play as they change the unfolding landscape in the presence and absence of NC. These data will contribute to understanding the TAR hairpin stability and provide insights for new HIV drug therapy