Nanomanipulation of single RNA molecules by optical tweezers.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Combining temperature and force to study folding of an RNA hairpin.

RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.

Analysis of diffuse K+ and Mg2+ ion binding to a two-base-pair kissing complex by single-molecule mechanical unfolding.

The folding and stability of RNA tertiary interactions depend critically on cationic conditions. It is usually difficult, however, to isolate such effects on tertiary interactions from those on the entire RNA. By manipulating conformations of single RNA molecules using optical tweezers, we distinguished individual steps of breaking and forming of a two-base-pair kissing interaction from those of secondary folding. The binding of metal ions to the small tertiary structure appeared to be saturable with an apparent Kd of 160 mM for K(+) and 1.5 mM for Mg(2+). The kissing formation was estimated to be associated with binding of ~2-3 diffuse K(+) or Mg(2+) ions. At their saturated binding, Mg(2+) provided ~3 kcal/mol more stabilizing energy to the structure than K(+). Furthermore, the cations change the unkissing forces significantly more than the kissing ones. For example, the presence of Mg(2+) ions increased the average unkissing force from 21 pN to 44 pN, surprisingly high for breaking merely two base pairs; in contrast, the mean kissing force was changed by only 4.5 pN. Interestingly, the differential salt effects on the transition forces were not caused by different changes in the height of the kinetic barriers but were instead attributed to how different molecular structures respond to the applied force. Our results showed the importance of diffuse cation binding to the stability of tertiary interaction and demonstrated the utility of mechanical unfolding in studying tertiary interactions.

The essential role of stacking adenines in a two-base-pair RNA kissing complex.

In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5'-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.

Mechanical unfolding of two DIS RNA kissing complexes from HIV-1.

An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg 2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 +/- 4 to 46 +/- 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of deltaX(++), which is the distance from the folded structure to the unfolding transition state. The deltaX(++) of Mal is approximately 0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg 2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg 2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.

How RNA unfolds and refolds.

Understanding how RNA folds and what causes it to unfold has become more important as knowledge of the diverse functions of RNA has increased. Here we review the contributions of single-molecule experiments to providing answers to questions such as: How much energy is required to unfold a secondary or tertiary structure? How fast is the process? How do helicases unwind double helices? Are the unwinding activities of RNA-dependent RNA polymerases and of ribosomes different from other helicases? We discuss the use of optical tweezers to monitor the unfolding activities of helicases, polymerases, and ribosomes, and to apply force to unfold RNAs directly. We also review the applications of fluorescence and fluorescence resonance energy transfer to measure RNA dynamics.

Real-time control of the energy landscape by force directs the folding of RNA molecules.

The rugged folding-energy landscapes of RNAs often display many competing minima. How do RNAs discriminate among competing conformations in their search for the native state? By using optical tweezers, we show that the folding-energy landscape can be manipulated to control the fate of an RNA: individual RNA molecules can be induced into either native or misfolding pathways by modulating the relaxation rate of applied force and even be redirected during the folding process to switch from misfolding to native folding pathways. Controlling folding pathways at the single-molecule level provides a way to survey the manifold of folding trajectories and intermediates, a capability that previously was available only to theoretical studies.

Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results.

Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1-10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010-3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.

Determination of thermodynamics and kinetics of RNA reactions by force.

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

Unusual mechanical stability of a minimal RNA kissing complex.

By using optical tweezers, we have investigated the mechanical unfolding of a minimal kissing complex with only two G.C base pairs. The loop-loop interaction is exceptionally stable; it is disrupted at forces ranging from 7 to 30 pN, as compared with 14-20 pN for unfolding hairpins of 7 and 11 bp. By monitoring unfolding/folding trajectories of single molecules, we resolved the intermediates, measured their rate constants, and pinpointed the rate-limiting steps. The two hairpins unfold only after breaking the intramolecular kissing interaction, and the kissing interaction forms only after the folding of the hairpins. At forces that favor the unfolding of the hairpins, the entire RNA structure is kinetically stabilized by the kissing interaction, and extra work is required to unfold the metastable hairpins. The strong mechanical stability of even a minimal kissing complex indicates the importance of such loop-loop interactions in initiating and stabilizing RNA dimers in retroviruses.

Probing the mechanical folding kinetics of TAR RNA by hopping, force-jump, and force-ramp methods.

Mechanical unfolding and refolding of single RNA molecules have previously been observed in optical traps as sudden changes in molecular extension. Two methods have been traditionally used: "force-ramp", with the applied force continuously changing, and "hopping". In hopping experiments the force is held constant and the molecule jumps spontaneously between two different states. Unfolding/refolding rates are measured directly, but only over a very narrow range of forces. We have now developed a force-jump method to measure the unfolding and refolding rates independently over a wider range of forces. In this method, the applied force is rapidly stepped to a new value and either the unfolding or refolding event is monitored through changes in the molecular extension. The force-jump technique is compared to the force-ramp and hopping methods by using a 52-nucleotide RNA hairpin with a three-nucleotide bulge, i.e., the transactivation response region RNA from the human immunodeficiency virus. We find the unfolding kinetics and Gibbs free energies obtained from all three methods to be in good agreement. The transactivation response region RNA hairpin unfolds in an all-or-none two-state reaction at any loading rate with the force-ramp method. The unfolding reaction is reversible at small loading rates, but shows hysteresis at higher loading rates. Although the RNA unfolds and refolds without detectable intermediates in constant-force conditions (hopping and force-jump), it shows partially folded intermediates in force-ramp experiments at higher unloading rates. Thus, we find that folding of RNA hairpins can be more complex than a simple single-step reaction, and that application of several methods can improve understanding of reaction mechanisms.

Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity.

TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.

Using hetero-11-mers composed of wild type and mutant subunits to study tryptophan binding to TRAP and its role in activating RNA binding.

Expression of genes involved in tryptophan metabolism in Bacillus subtilis is regulated by the TRAP protein in response to changes in l-tryptophan levels. TRAP binding to several RNA targets that contain between 9 and 11 (G/U)AG repeats regulates transcription and/or translation of these genes. TRAP consists of 11 identical subunits and is activated to bind RNA by binding up to 11 molecules of tryptophan. To investigate the mechanism by which tryptophan binding activates TRAP, we generated hetero-11-mers containing different proportions of subunits from wild type (WT) TRAP that bind tryptophan and from a mutant TRAP (Thr(25) to Ala) defective in tryptophan binding. Studies of these hetero-11-mers show that tryptophan-binding sites created from active subunits bind tryptophan with similar affinity to those in WT homo-11-mers, whereas sites containing the T25A substitution do not bind tryptophan. Hetero-11-mers with very few (one or two) bound tryptophans show only 10-fold lower affinity than WT TRAP for an RNA with 11 GAG repeats, whereas TRAP with no bound tryptophan shows no detectable binding to this RNA. We also demonstrate that tryptophan binding induces a conformational change in TRAP in the vicinity of the RNA-binding site, suggesting a possible mechanism for activation of RNA binding.

Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP.

TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats. TRAP is composed of 11 identical subunits arranged symmetrically in a ring. The secondary structure of the protein consists entirely of antiparallel beta-sheets, beta-turns, and loops. We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride. Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers. Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11-mers by denaturation followed by subunit mixing renaturation. This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding. Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.