Search

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Forster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

Despite their importance in biology and use in nanotechnology, the elastic behavior of nucleic acids on "ultrashort" (15 nt) length scales remains poorly understood. Here, we use optical tweezers combined with fluorescence imaging to observe directly the hybridization of oligonucleotides (7-12 nt) to a complementary strand under tension and to measure the difference in end-to-end extension between the single-stranded and duplex states. Data are consistent with long-polymer models at low forces (8 pN) but smaller than predicted at higher forces (8 pN), the result of the sequence-dependent duplex edge effects.

Engineering superenzyme function Understanding how protein domains and subunits operate is critical for engineering novel functions into proteins. Arslan et al. introduced intramolecular crosslinks between two domains of the Escherichia coli helicase Rep, which unwinds DNA. By inserting linkers of different lengths, the domains can be held either “open” or “closed.” The closed conformation activates the helicase, but it can also generate super-helicases capable of unzipping long stretches of DNA at high speed and with considerable force. Comstock et al. used optical tweezers and fluorescence microscopy to simultaneously measure the structure and function of the bacterial helicase UvrD. They monitored its DNA winding and unwinding activity and its shape during these activities. The motor domain also has a “closed” conformation during DNA unwinding and switches to a reversed “open” conformation during the zipping-up interaction. Science , this issue p. 344 and p. 352

Helicases are a diverse set of enzymes that play an essential role in genome maintenance by converting chemical energy into the mechanical work of unzipping nucleic acid duplexes. E. coli UvrD is a prototypical superfamily 1 helicase involved in methyl-directed mismatch repair and nucleotide excision repair. Recent measurements have shown that it is able to both unwind and rezip the DNA duplex. Structural, ensemble kinetic, and single-molecule studies have provided insights into the unwinding mechanism of this helicase, but there has not yet been a direct observation of the individual motor steps involved in the process. Here, we use high-resolution optical tweezers to observe directly the stepping behavior of UvrD at a single-molecule level as it unwinds and rezips a DNA hairpin. Interestingly, we measure a step size of 3-4 base pairs for both unwinding and rezipping activities. Furthermore, our analysis of the stepping kinetics indicates that a single rate-limiting step governs the process, which is consistent with ensemble studies measuring an unwinding step size of 4-5 base pairs per rate-limiting step. However, previous studies have determined that 1 ATP molecule is hydrolyzed per base pair translocated, and would predict 3-4 rate-limiting steps per unwinding step. Our results therefore suggest a mechanism whereby UvrD rapidly unwinds 3-4 base pairs, and then must slowly “reset” itself in order to continue its unwinding cycle.

The hybridization of short oligonucleotides plays a critical role in many biological systems, from DNA replication to gene silencing. Despite extensive studies, details on the mechanism of this process on the shortest length scale (∼10 bp) remain poorly understood. We use high-resolution optical tweezers with simultaneous fluorescence microscopy to investigate the hybridization of single oligonucleotides under tension1. We measure the change in end-to-end extension upon annealing and melting as well as the unbinding kinetics of short (7-12 bp), fluorescently labeled oligonucleotides hybridizing to a complementary DNA sequence tethered between trapped beads. Our results allow us to construct an energy landscape of oligonucleotide hybridization along well-defined reaction coordinates. Interestingly, our measurement of the distance to the transition state for DNA melting as a function of oligonucleotide length suggests that the extension of the transition state matches that of a state with 6 bp hybridized. Lastly, we find that the change in extension upon hybridization deviates from extensible-wormlike-chain behavior at forces >10 pN. We discuss possible models to explain these results.1. M. J. Comstock, Taekjip Ha, and Y. R. Chemla, Nat. Meth. 8, 4 (2011).

We present single-molecule high-resolution optical trapping measurements of RNA unwinding by the Ski2-like nuclear helicase Mtr4p both alone and within the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex. RNA helicases such as Mtr4p play key roles in RNA metabolism. Mtr4p is thought to assist RNA degradation by the exosome presumably by unwinding RNA structures. In addition, the TRAMP complex appends short 3′ poly(A) tails to RNA meant for degradation by the exosome. Mtr4p within the TRAMP complex plays a crucial role in regulating the polyadenylation activity, limiting the poly(A) tail length. The unwinding activity of Mtr4p is required and indeed within the TRAMP complex, Mtr4p unwinds duplex RNA 10x faster than when alone. However, the dynamic details of the RNA unwinding activity and interaction of Mtr4p and TRAMP and indeed the Ski2-like family of RNA helicases are unknown. We have performed high-resolution RNA hairpin unwinding experiments for both Mtr4p and TRAMP. We find that Mtr4p unwinds RNA duplexes in a single or multiple bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4p remains stably bound after unwinding the duplex, preventing re-annealing. TRAMP unwinding experiments reveal a faster molecular recognition by the Mtr4p helicase when in complex as well as enhanced RNA duplex unwinding dynamics. Combined high-resolution trapping and fluorescence experiments underway may reveal how the competition within TRAMP between the 3′-to-5′ Mtr4p helicase and the 5′-to-3′ Trf4p polymerase is resolved dynamically.

We present single-molecule in vitro measurements of the DNA unwinding activity of the Superfamily 1 UvrD helicase that reveal directly how helicase stoichiometry and conformation correspond to motor activity. Although a single UvrD molecule can translocate along ssDNA, it has been postulated that only a dimer is capable of unwinding duplex DNA. UvrD is known to exhibit two different conformations: the ‘closed' and ‘open' orientation of the 2B regulatory subdomain. The function of these conformations has remained elusive. using a new instrument combining high-resolution optical tweezers with simultaneous single-molecule fluorescence microscopy (Comstock, Ha, and Chemla, Nature Methods 2011), we obtain an unprecedented detailed view of helicase motor dynamics by recording single base pair-scale DNA unwinding activity (optical tweezers) simultaneously with helicase stoichiometry and conformation (fluorescence). We observe two classes of DNA unwinding by UvrD: long distance continuous unwinding (∼100 bp) and repetitive, non-processive (∼12 bp) unwinding. We determine directly the stoichiometry of UvrD molecules participating in unwinding by quantifying the fluorescence intensity using singly-labeled UvrD molecules. We observe that pairs of UvrD molecules are required for long distance unwinding but individual molecules exhibit limited non-processive unwinding activity. Furthermore, we observe directly the conformation of the 2B subdomain of individual UvrD molecules during unwinding by measuring the fluorescence of FRET-labeled proteins. We observe that UvrD is in the ‘closed' conformation during DNA unwinding but surprisingly switches to the ‘open' conformation when UvrD translocates on the opposing ssDNA strand away from the junction with the DNA junction re-annealing behind it. These measurements may explain UvrD helicase's remarkable ability to switch translocation strands. We hypothesize that the UvrD 2B subdomain acts as a switch that controls DNA unwinding vs. re-annealing.