Julie L. Fiore

Entropic origin of Mg2+-facilitated RNA folding

Mg2+ is essential for the proper folding and function of RNA, though the effect of Mg2+ concentration on the free energy, enthalpy, and entropy landscapes of RNA folding is unknown. This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop−receptor tertiary interaction as a function of [Mg2+]. These measurements yield the barrier and standard state enthalpies, entropies, and free energies for an RNA tertiary transition, in particular, revealing the thermodynamic origin of [Mg2+]-facilitated folding. Surprisingly, these studies reveal that increasing [Mg2+] promotes tetraloop–receptor interaction by reducing the entropic barrier () and the overall entropic penalty () for docking, with essentially negligible effects on both the activation enthalpy () and overall exothermicity (). These observations contrast with the conventional notion that increasing [Mg2+] facilitates folding by minimizing electrostatic repulsion of opposing RNA helices, which would incorrectly predict a decrease in and with [Mg2+]. Instead we propose that higher [Mg2+] can aid RNA folding by decreasing the entropic penalty of counterion uptake and by reducing disorder of the unfolded conformational ensemble.

An RNA folding motif: GNRA tetraloop–receptor interactions

Nearly two decades after Westhof and Michel first proposed that RNA tetraloops may interact with distal helices, tetraloop–receptor interactions have been recognized as ubiquitous elements of RNA tertiary structure. The unique architecture of GNRA tetraloops (N=any nucleotide, R=purine) enables interaction with a variety of receptors, e.g., helical minor grooves and asymmetric internal loops. The most common example of the latter is the GAAA tetraloop–11 nt tetraloop receptor motif. Biophysical characterization of this motif provided evidence for the modularity of RNA structure, with applications spanning improved crystallization methods to RNA tectonics. In this review, we identify and compare types of GNRA tetraloop–receptor interactions. Then we explore the abundance of structural, kinetic, and thermodynamic information on the frequently occurring and most widely studied GAAA tetraloop–11 nt receptor motif. Studies of this interaction have revealed powerful paradigms for structural assembly of RNA, as well as providing new insights into the roles of cations, transition states and protein chaperones in RNA folding pathways. However, further research will clearly be necessary to characterize other tetraloop–receptor and long-range tertiary binding interactions in detail – an important milestone in the quantitative prediction of free energy landscapes for RNA folding.

Monovalent and divalent promoted GAAA tetraloop-receptor tertiary interactions from freely diffusing single-molecule studies.

Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg(2+) and Na(+) concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na(+)] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 +/- 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg(2+)] levels, which agrees quantitatively with the 32 +/- 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the K(D) value and Hill coefficient for [Mg(2+)]-dependent docking decrease significantly for [Na(+)] = 25 mM vs. 125 mM, indicating Mg(2+) and Na(+) synergy in the RNA folding process.

The Role of Counterion Valence and Size in GAAA Tetraloop–Receptor Docking/Undocking Kinetics

For RNA to fold into compact, ordered structures, it must overcome electrostatic repulsion between negatively charged phosphate groups by counterion recruitment. A physical understanding of the counterion-assisted folding process requires addressing how cations kinetically and thermodynamically control the folding equilibrium for each tertiary interaction in a full-length RNA. In this work, single-molecule FRET (fluorescence resonance energy transfer) techniques are exploited to isolate and explore the cation-concentration-dependent kinetics for formation of a ubiquitous RNA tertiary interaction, that is, the docking/undocking of a GAAA tetraloop with its 11-nt receptor. Rate constants for docking (k(dock)) and undocking (k(undock)) are obtained as a function of cation concentration, size, and valence, specifically for the series Na(+), K(+), Mg(2+), Ca(2+), Co(NH(3))(6)(3+), and spermidine(3+). Increasing cation concentration acceleratesk(dock)dramatically but achieves only a slight decrease in k(undock). These results can be kinetically modeled using parallel cation-dependent and cation-independent docking pathways, which allows for isolation of the folding kinetics from the interaction energetics of the cations with the undocked and docked states, respectively. This analysis reveals a preferential interaction of the cations with the transition state and docked state as compared to the undocked RNA, with the ion-RNA interaction strength growing with cation valence. However, the corresponding number of cations that are taken up by the RNA upon folding decreases with charge density of the cation. The only exception to these behaviors is spermidine(3+), whose weaker influence on the docking equilibria with respect to Co(NH(3))(6)(3+) can be ascribed to steric effects preventing complete neutralization of the RNA phosphate groups.

Multi-kilohertz repetition rate Ti:sapphire amplifier based on down-chirped pulse amplification

We present a novel ultrafast multipass laser amplifier design optimized for sub-millijoule output energy and capable of being operated at repetition rates exceeding 40 kHz. This ti:sapphire based system makes use of a grism based stretcher, a cryogenically cooled ti:sapphire crystal and an astigmatically compensated multipass amplifier design that allows for pumping with significantly lower pump pulse energies than has been demonstrated to date. We also make use of the downchirped pulse amplification scheme to minimize loss in the pulse compression process. Preliminary experiments demonstrate an output pulse energy of 290 muJ at 10 kHz and 270 muJ at 15 kHz with a pulse duration of 36 fs.

Suppressing nonspecific adsorption of proteins on the single-molecular level

Avoiding nonspecific surface adsorption is a crucial and often challenging issue in many single-molecule studies and analytical applications. In this work, we investigated glass surfaces coated with cross-linking star-shaped polyethylene glycol (4-arm PEG) and demonstrated that this coating can be used for effective suppression of nonspecific protein binding, such as streptavidin. Single-molecule fluorescence images show that only a few molecules remain nonspecifically bound to surfaces treated with protein after sufficient rinsing, i.e. less than to a state-of-the-art BSA coating. Furthermore, different applications for star-shaped PEG-passivated surfaces are shown.

The Role of Bulges and Hinges in RNA Folding

Long-range tertiary interactions govern RNA structural assembly, which is a critical step toward RNA biological functionality. Thereby, a universal strategy has emerged for global conformational change; flexible junctions enable unpaired nucleotides to act as beacons between helical regions. Bulges, for example, are versatile secondary structural elements implicated in helix recognition and packing. The P4-P6 domain of the Tetrahymena ribozyme utilizes this folding strategy by hinging to form two inter-helical tertiary contacts, the adenine-rich (A-rich) bulge and the tetraloop-tetraloop receptor interactions. To explore the kinetic and thermodynamic properties of tertiary contact formation, we probe the P4-P6 domain hinging and ribose zippering that forms the A-rich bulge interaction using single-molecule FRET methods. We obtain the docking and undocking rates of the A-rich bulge and P4 helix as function of cation concentration and temperature. Docking is accelerated and undocking decelerated by Mg2+. In spite of rapid docking at high [Mg2+] (kdock = 20 ± 2 s−1), the A-rich bulge interaction is only marginally stable (Kdock = 1.2 ± 0.1). These results support that the role of the A-rich bulge is to kinetically direct P4-P6 domain folding while thermodynamic stability is added through the tetraloop-receptor interaction. Formation of the A-rich bulge contact shows specificity for divalent cations, with a preference for Mg2+ as anticipated from the Mg2+ coordination observed in structural data. A significant kinetic heterogeneity is characterized; only 50% of the molecules exhibit efficient folding at high [Mg2+]. Mutations of the A-rich bulge construct reveal a crucial role of the P4-P6 secondary architecture in enabling the A-rich bulge contact.