Session L10: Focus Session: Single Molecule Biophysics and Chemical Physics IV

A Kinetic and Thermodynamic Perspective of RNA Folding Transition States

Structural assembly is critical to RNA biological functionality. However, an energetic and molecular description of how RNA folds pales in comparison to the understanding of protein folding. To gain molecular insight into the energetic barriers along RNA folding pathways, we explore the transition states for an elementary folding step by extracting the enthalpy and entropy changes associated with forming a single ubiquitous tertiary interaction. By combining single-molecule fluorescence resonance energy transfer methods with temperature variation, we measure the temperature-dependent docking and undocking rate constants of an intramolecular tetraloop--receptor motif. Tetraloop--receptor RNA folds through an early transition state with an entropic barrier. The net reaction is exothermic and entropically costly with a small favorable free energy change at 1 mM Mg$^{2+}$. Exploring the enthalpy and entropy changes along the reaction coordinate at varying [Mg$^{2+}$], reveals that Mg$^{2+}$-enhanced folding originates from a reduction in the entropic barrier and net cost of folding that outweighs unfavorable enthalpic effects.   

Kinetic Studies of Lysine Riboswitch Folding Using Single-Molecule FRET

Riboswitches regulate gene expression through conformational changes induced by metabolite binding. This regulation of gene expression depends on the kinetics of metabolite binding and structural changes. Therefore, an understanding of these dynamics is crucial to developing a compete knowledge of riboswitch functionality. To probe the binding of a metabolite and subsequent folding, a metabolite-binding domain of the \textit{Bacillus subtilis} lysine riboswitch was transcribed and hybridized to a fluorescent-labeled RNA strand, which allows FRET monitoring of ligand-induced conformational changes. The RNA construct was studied using single-molecule FRET methods that allowed for characterization of the folding dynamics. In the presence of lysine, we observed two states, of which the relative populations are perturbed by lysine concentration. We measured the folding and unfolding rates of the inter-conversion between these states. We also observe that [Mg$^{2+}$] affects the lysine-free conformation and the lysine sensitivity of the riboswitch.   

Kinetics of single DNA hairpin dissociation

Over the past decade, groups have used a variety of single molecule techniques to study the unfolding and unbinding of nucleic acids, proteins and other biomolecules. While some experiments on the dissociation of nucleic acids find exponential lifetime distributions, as expected for a process governed by a single rate-limiting pathway, other experiments find nonexponential lifetime distributions. In our work, we address this discrepancy by probing the force dependence of a single DNA hairpin under thermal dissociation. We use a scanning line optical tweezers instrument to measure the bound lifetimes of two DNA-coated microspheres under negligible applied tension. The two microspheres share a user-specified potential along the scan direction and are strongly confined in the perpendicular dimensions. The trapping laser intensity is modulated synchronously with a resonant scanning mirror to null all optical contributions to the pair interaction potential near contact. In addition, the laser polarization can be rotated to produce a continuously adjustable optical repulsion, allowing the instrument to double as a passive force clamp over a modest range of applied tensions. This unique experimental approach allows us to investigate many of the proposed explanations for nonexponential kinetics in nucleic acid dissociation that have, until now, been difficult to isolate.   

Single-molecule FRET studies of RNA folding and catalysis

Single-molecule FRET has been applied to the folding of branched DNA and RNA structures, and their dynamics. We find that FRET efficiencies from single junctions encapsulated within phospholipid vesicles are more reliable than those from ensemble measurements, and a valuable source of structural data. We have shown that both cyanine fluorophores are substantially stacked upon the end of double-stranded DNA or RNA when attached at the 5'-terminus. This leads to a significant orientation dependence of FRET efficiency that is incompletely averaged by lateral motion of the fluorophores. Using a series of DNA and RNA duplexes we have shown that the orientation dependence leads to a pronounced modulation of efficiency as a function of helix length; these data unequivocally establish that F\"orster transfer obeys the orientation dependence as expected for a dipole-dipole interaction Ignoring this effect can lead to significant errors in distance determination. However, a full understanding of the orientation dependence could greatly extend the use of FRET measurements to provide both accurate distance and angular information.   

Laser-Assisted Single Molecule Refolding

In vivo, many RNA molecules can adopt multiple conformations depending on their biological context such as the HIV Dimerization Initiation Sequence (DIS) or the DsrA RNA in bacteria. It is quite common that the initial interaction between the two RNAs takes place via complementary unpaired regions, thus forming a so-called kissing complex. However, the exact kinetic mechanism by which the two RNA molecules reach the dimerized state is still not well understood. To investigate the refolding energy surface of RNA molecules, we have developed new technology based on the combination of single molecule spectroscopy with laser induced temperature jump kinetics, called Laser Assisted Single-molecule Refolding (LASR). LASR enables us to induce folding reactions of otherwise kinetically trapped RNAs at the single molecule level, and to characterize their folding landscape. LASR provides an exciting new approach to study molecular memory effects and kinetically trapped RNAs in general. LASR should be readily applicable to study DNA and protein folding as well.   

Localized Heating of Single Nucleic Acids using Infrared Light

To alleviate many of the shortcomings associated with bulk sample heating at the objective of a microscope, we have developed a system that uses a cw IR laser (1455 nm) to locally heat a small volume of water via absorption in the second overtone of the OH stretch. By focusing the IR laser onto the confocal volume of a single-molecule fluorescence microscope, we can rapidly and locally adjust the sample temperature. A resonantly absorbing thin water cell placed after the sample but before our detection system removes residual transmitted IR light, allowing for continuous observation of fluorescent molecules while heating. With time-correlated single-photon counting and the known temperature-dependent lifetime of Rhodamine B, we have calibrated the range of temperature achievable by IR laser heating to be from room temperature to greater than 90 \r{ }C. We apply this system to the melting of duplex nucleic acids tethered to a glass surface. Additionally, we demonstrate that the localized IR laser can function as a static heat source for temperature-dependent kinetic studies of RNA folding, which prove to be in excellent agreement with previous measurements using bulk stage heating.   

Confining individual DNA molecules in an axisymmetric entropy gradient

Many asymmetric and discontinuous confining environments have been used to study the properties of confined DNA. We have developed a unique method for studying DNA in micropipettes, resulting in a confining environment that is axisymmetric with a continuously changing entropy gradient. An applied electric field forces the chain into sub-micron confinement and fluorescence microscopy is used to track the effect of confinement on the entropy of individual DNA chains. Releasing the electric field, we probe the dynamics of the DNA chain in a continuously changing confinement, yielding a comprehensive study of the entropic force. This technique provides a novel method for studying the effect of polymer chain architecture on entropy. These architectures include knots in polymer chains, cyclic chains, or the presence of histones amongst DNA molecules.   

The Effect of the Refractive Index of the Medium in Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a useful tool to study diffusional motion in liquids as it measures resident time of a dye molecule in a small excitation volume made by confocal microscopy. Some reports recently predicted that the measurement result of FCS is affected sensitively by the refractive index of liquid medium. To check for this possibility, several liquids having the same viscosity values but different refractive indices were chosen to dissolve dye molecules. The change in the observed diffusion coefficients in solutions having the same viscosity value manifests that care needs to be taken in the common practice of using sucrose to change the viscosity in the FCS experiment.   

Theory for Frequency-Resolved Photon Emission Statistics in Single-Molecule Fluorescence Spectroscopy

We derive the moment generating function for photon emission from a single molecule driven by laser excitation. The frequencies of the fluoresced photons are explicitly considered. Calculations were performed for the case of a two level dye molecule, showing that measured photon statistics will display a strong and nonintuitive dependence of detector bandwidth. Moreover it is demonstrated that the anti-bunching phenomenon associated with negative values of Mandel's Q parameter, results from correlations between photons with well separated frequencies.