Correction to “Single-Molecule FRET Studies of HIV TAR-DNA Hairpin Unfolding Dynamics”

We directly measure the dynamics of the HIV trans-activation response (TAR)−DNA hairpin with multiple loops using single-molecule Förster resonance energy transfer (smFRET) methods. Multiple FRET states are identified that correspond to intermediate melting states of the hairpin. The stability of each intermediate state is calculated from the smFRET data. The results indicate that hairpin unfolding obeys a “fraying and peeling” mechanism, and evidence for the collapse of the ends of the hairpin during folding is observed. These results suggest a possible biological function for hairpin loops serving as additional fraying centers to increase unfolding rates in otherwise stable systems. The experimental and analytical approaches developed in this article provide useful tools for studying the mechanism of multistate DNA hairpin dynamics and of other general systems with multiple parallel pathways of chemical reactions. ■ INTRODUCTION Themelting and annealing of DNA hairpins are essential in many biological processes such as replication, transcription, recombination, gene expression, and DNA transposition for both prokaryotic and eukaryotic systems. Furthermore, hairpins with multiple loops are known to play specific roles in viral replication. An important example is the human immunodeficiency virus-1 (HIV-1) trans-activation response region (TAR) hairpin. The TAR sequence is remarkably well conserved among HIV isolates, indicating a strong selection pressure to maintain its structure. Thus, the TAR hairpin is of therapeutic interest. The TAR−RNA hairpin and its complement, TAR− DNA hairpin, are involved in several crucial steps in the viral life cycle. The TAR hairpin has four bulges, which have been found to be critical to the biological function of the TAR sequence because they determine the hairpin unfolding/folding dynamics. As a general topic, understanding hairpin dynamics is further motivated by the advent of therapeutics with aptamers, which are small RNA and DNA molecules that often form single or multiloop hairpin conformations. In order to understand the molecular-scale dynamics of DNA/ RNA hairpins, hairpins have been studied using technologies such as temperature-jump, optical trap, single-molecule fluorescence resonance energy transfer (smFRET), and combinations of spectroscopic techniques. However, these hairpin structures usually have one single loop connecting a stem region of several base pairs (Figure 1a). It is generally understood that the unfolding/folding rates of such simple DNA hairpins are dependent on the binding energy of the hairpin, the diffusion rate of the two ends of the stem followed by nucleation, and the propagation of base pairing. This process yields folding times that range from milliseconds to microseconds, depending on the sequence length and base composition. smFRET is particularly suited to this study due to its wide applications in studying the single-molecule dynamics of nucleic acids. For DNA melting (unfolding), a “fraying and peeling mechanism” has been predicted, and for annealing (folding) Received: July 15, 2014 Revised: September 23, 2014 Published: September 25, 2014 Figure 1. Schematic of proposed examples of unfolding/folding routes of (a, b) model DNA hairpins and (c) the TAR−DNA hairpin with two dyes Cy3 and Cy5 labeled to the ends. Urea molecules within the solution are shown, and the double helix is not shown for easier demonstration. Article