Probing and perturbing riboswitch folding using a fluorescent base analogue.

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ1 riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ1 . We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ1 at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ1 concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch.

Disentangling contact and ensemble epistasis in a riboswitch.

Mutations introduced into macromolecules often exhibit epistasis, where the effect of one mutation alters the effect of another. Knowing the mechanisms that lead to epistasis is important for understanding how macromolecules work and evolve, as well as for effective macromolecular engineering. Here, we investigate the interplay between "contact epistasis" (epistasis arising from physical interactions between mutated residues) and "ensemble epistasis" (epistasis that occurs when a mutation redistributes the conformational ensemble of a macromolecule, thus changing the effect of the second mutation). We argue that the two mechanisms can be distinguished in allosteric macromolecules by measuring epistasis at differing allosteric effector concentrations. Contact epistasis manifests as nonadditivity in the microscopic equilibrium constants describing the conformational ensemble. This epistatic effect is independent of allosteric effector concentration. Ensemble epistasis manifests as nonadditivity in thermodynamic observables-such as ligand binding-that are determined by the distribution of ensemble conformations. This epistatic effect strongly depends on allosteric effector concentration. Using this framework, we experimentally investigated the origins of epistasis in three pairwise mutant cycles introduced into the adenine riboswitch aptamer domain by measuring ligand binding as a function of allosteric effector concentration. We found evidence for both contact and ensemble epistasis in all cycles. Furthermore, we found that the two mechanisms of epistasis could interact with each other. For example, in one mutant cycle we observed 6 kcal/mol of contact epistasis in a microscopic equilibrium constant. In that same cycle, the maximum epistasis in ligand binding was only 1.5 kcal/mol: shifts in the ensemble masked the contribution of contact epistasis. Finally, our work yields simple heuristics for identifying contact and ensemble epistasis based on measurements of a biochemical observable as a function of allosteric effector concentration.

Base-Stacking Heterogeneity in RNA Resolved by Fluorescence-Detected Circular Dichroism Spectroscopy.

RNA plays a critical role in many biological processes, and the structures it adopts are intimately linked to those functions. Among many factors that contribute to RNA folding, van der Waals interactions between adjacent nucleobases stabilize structures in which the bases are stacked on top of one another. Here, we utilize fluorescence-detected circular dichroism spectroscopy (FDCD) to investigate base-stacking heterogeneity in RNA labeled with the fluorescent adenine analogue 2-aminopurine (2-AP). Comparison of standard (transmission-detected) CD and FDCD spectra reveals that in dinucleotides, 2-AP fluorescence is emitted almost exclusively by unstacked molecules. In a trinucleotide, some fluorescence is emitted by a population of stacked and highly quenched molecules, but more than half originates from a minor ∼10% population of unstacked molecules. The combination of FDCD and standard CD measurements reveals the prevalence of stacked and unstacked conformational subpopulations as well as their relative fluorescence quantum yields.

An anionic ligand snap-locks a long-range interaction in a magnesium-folded riboswitch.

The archetypical transcriptional crcB fluoride riboswitch from Bacillus cereus is an intricately structured non-coding RNA element enhancing gene expression in response to toxic levels of fluoride. Here, we used single molecule FRET to uncover three dynamically interconverting conformations appearing along the transcription process: two distinct undocked states and one pseudoknotted docked state. We find that the fluoride anion specifically snap-locks the magnesium-induced, dynamically docked state. The long-range, nesting, single base pair A40-U48 acts as the main linchpin, rather than the multiple base pairs comprising the pseudoknot. We observe that the proximally paused RNA polymerase further fine-tunes the free energy to promote riboswitch docking. Finally, we show that fluoride binding at short transcript lengths is an early step toward partitioning folding into the docked conformation. These results reveal how the anionic fluoride ion cooperates with the magnesium-associated RNA to govern regulation of downstream genes needed for fluoride detoxification of the cell.

Versatile transcription control based on reversible dCas9 binding.

The ability to control transcription in a time-dependent manner in vitro promises numerous applications in molecular biology and nanotechnology. Here we demonstrate an approach that enables precise, independent control over the production of multiple RNA transcripts in vitro using single guide RNA (sgRNA)-directed transcription blockades by catalytically dead Streptococcus pyogenes CRISPR-Cas9 enzyme (dCas9). We show that when bound to a DNA template, the dCas9:sgRNA complex forms a robust blockade to transcription by RNA polymerases (RNAPs) from bacteriophages SP6, T3, and T7 (>99.5% efficiency), and a partial blockade to transcription by Escherichia coli RNAP (∼70% efficiency). We find that all three bacteriophage RNAPs dissociate from the DNA template upon encountering the dCas9 blockade, while E. coli RNAP stays bound for at least the 90-min duration of our experiments. The blockade maintains >95% efficiency when four mismatches are introduced into the 5' end of the sgRNA target sequence. Notably, when using such a mismatched blockade, production of specific RNA species can be activated on demand by addition of a double-stranded competitor DNA perfectly matching the sgRNA. This strategy enables the independent production of multiple RNA species in a temporally controlled fashion from the same DNA template, demonstrating a new approach for transcription control.

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing.

Numerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

Life under the Microscope: Single-Molecule Fluorescence Highlights the RNA World.

The emergence of single-molecule (SM) fluorescence techniques has opened up a vast new toolbox for exploring the molecular basis of life. The ability to monitor individual biomolecules in real time enables complex, dynamic folding pathways to be interrogated without the averaging effect of ensemble measurements. In parallel, modern biology has been revolutionized by our emerging understanding of the many functions of RNA. In this comprehensive review, we survey SM fluorescence approaches and discuss how the application of these tools to RNA and RNA-containing macromolecular complexes in vitro has yielded significant insights into the underlying biology. Topics covered include the three-dimensional folding landscapes of a plethora of isolated RNA molecules, their assembly and interactions in RNA-protein complexes, and the relation of these properties to their biological functions. In all of these examples, the use of SM fluorescence methods has revealed critical information beyond the reach of ensemble averages.

Soft Interactions with Model Crowders and Non-canonical Interactions with Cellular Proteins Stabilize RNA Folding.

Living cells contain diverse biopolymers, creating a heterogeneous crowding environment, the impact of which on RNA folding is poorly understood. Here, we have used single-molecule fluorescence resonance energy transfer to monitor tertiary structure formation of the hairpin ribozyme as a model to probe the effects of polyethylene glycol and yeast cell extract as crowding agents. As expected, polyethylene glycol stabilizes the docked, catalytically active state of the ribozyme, in part through excluded volume effects; unexpectedly, we found evidence that it additionally displays soft, non-specific interactions with the ribozyme. Yeast extract has a profound effect on folding at protein concentrations 1000-fold lower than found intracellularly, suggesting the dominance of specific interactions over volume exclusion. Gel shift assays and affinity pull-down followed by mass spectrometry identified numerous non-canonical RNA-binding proteins that stabilize ribozyme folding; the apparent chaperoning activity of these ubiquitous proteins significantly compensates for the low-counterion environment of the cell.

Electric Dipole Transition Moments and Solvent-Dependent Interactions of Fluorescent Boron-Nitrogen Substituted Indole Derivatives.

Fluorescent analogues of the indole side chain of tryptophan can be useful spectroscopic probes of protein-protein and protein-DNA interactions. Here we present linear dichroism and solvent-dependent spectroscopic studies of two fluorescent analogues of indole, in which the organic C═C unit is substituted with the isosteric inorganic B-N unit. We studied the so-called "external" BN indole, which has C2v symmetry, and the "fused" BN indole with Cs symmetry. We performed a combination of absorption and fluorescence spectroscopy, ultraviolet linear dichroism (UV-LD) in stretched poly(ethylene) (PE) films, and quantum chemical calculations on both BN indole compounds. Our measurements allowed us to characterize the degree of alignment for both molecules in stretched PE films. We thus determined the orientations and magnitudes of the two lowest energy electric dipole transition moments (EDTMs) for external BN indole, and the two lowest energy EDTMs for fused BN indole within the 30 000-45 000 cm(-1) spectral range. We compared our experimental results to those of quantum chemical calculations using standard density functional theory (DFT). Our theoretical predictions for the low-energy EDTMs are in good agreement with our experimental data. The absorption and fluorescence spectra of the external and the fused BN indoles are sensitive to solvent polarity. Our results indicate that the fused BN indole experiences much greater solvation interactions with polar solvents than does the external BN indole.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.

Coherent two-dimensional photocurrent spectroscopy in a PbS quantum dot photocell.

Recently there has been growing interest in the role of coherence in electronic dynamics. Coherent multidimensional spectroscopy has been used to reveal coherent phenomena in numerous material systems. Here we utilize a recent implementation of coherent multidimensional spectroscopy--two-dimensional photocurrent spectroscopy--in which we detect the photocurrent from a PbS quantum dot photocell resulting from its interactions with a sequence of four ultrafast laser pulses. We observe sub-picosecond evolution of two-dimensional spectra consistent with multiple exciton generation. Moreover, a comparison with two-dimensional fluorescence spectra of the quantum dots demonstrates the potential of two-dimensional photocurrent spectroscopy to elucidate detailed origins of photocurrent generating electronic state coherence pathways. Since the measurement is based on detecting the photocell current in situ, the method is well suited to study the fundamental ultrafast processes that affect the function of the device. This opens new avenues to investigate and implement coherent optimization strategies directly within devices.

Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update.

Toxicology is the highly interdisciplinary field studying the adverse effects of chemicals on living organisms. It requires sensitive tools to detect such effects. After their initial implementation during the 1990s, single-molecule fluorescence detection tools were quickly recognized for their potential to contribute greatly to many different areas of scientific inquiry. In the intervening time, technical advances in the field have generated ever-improving spatial and temporal resolution and have enabled the application of single-molecule fluorescence to increasingly complex systems, such as live cells. In this review, we give an overview of the optical components necessary to implement the most common versions of single-molecule fluorescence detection. We then discuss current applications to enzymology and structural studies, systems biology, and nanotechnology, presenting the technical considerations that are unique to each area of study, along with noteworthy recent results. We also highlight future directions that have the potential to revolutionize these areas of study by further exploiting the capabilities of single-molecule fluorescence microscopy.

Solution conformation of 2-aminopurine (2-AP) dinucleotide determined by ultraviolet 2D fluorescence spectroscopy (UV-2D FS).

We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) - a fluorescence-detected variation of 2D electronic spectroscopy - to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.

Entangled photon-pair two-dimensional fluorescence spectroscopy (EPP-2DFS).

We introduce a new method, called entangled photon-pair two-dimensional fluorescence spectroscopy (EPP-2DFS), to sensitively probe the nonlinear electronic response of molecular systems. The method incorporates a separated two-photon ('Franson') interferometer, which generates time-frequency-entangled photon pairs, into the framework of a fluorescence-detected 2D optical spectroscopic experiment. The entangled photons are temporally shaped and phase-modulated in the interferometer, and are used to excite a two-photon-absorbing (TPA) sample, whose excited-state population is selectively detected by simultaneously monitoring the sample fluorescence and the exciting fields. In comparison to 'classical' 2DFS techniques, major advantages of this scheme are the suppression of uncorrelated background signals, the enhancement of simultaneous time-and-frequency resolution, the suppression of diagonal 2D spectral features, and the enhancement and narrowing of off-diagonal spectral cross-peaks that contain information about electronic couplings. These effects are a consequence of the pure-state field properties unique to a parametric down-conversion light source, which must be included in the quantum mechanical description of the composite field-molecule system. We numerically simulate the EPP-2DFS observable for the case of an electronically coupled molecular dimer. The EPP-2DFS spectrum is greatly simplified in comparison to its classical 2D counterpart. Our results indicate that EPP-2DFS can provide previously unattainable resolution to extract model Hamiltonian parameters from electronically coupled molecular dimers.

Temperature-dependent conformations of a membrane supported zinc porphyrin tweezer by 2D fluorescence spectroscopy.

We studied the equilibrium conformations of a zinc porphyrin tweezer composed of two carboxylphenyl-functionalized zinc tetraphenyl porphyrin subunits connected by a 1,4-butyndiol spacer, which was suspended inside the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomes. By combining phase-modulation two-dimensional fluorescence spectroscopy (2D FS) with linear absorbance and fluorimetry, we determined that the zinc porphyrin tweezer adopts a mixture of folded and extended conformations in the membrane. By fitting an exciton-coupling model to a series of data sets recorded over a range of temperatures (17-85 °C) and at different laser center wavelengths, we determined that the folded form of the tweezer is stabilized by a favorable change in the entropy of the local membrane environment. Our results provide insights toward understanding the balance of thermodynamic factors that govern molecular assembly in membranes.

Electronic transition moments of 6-methyl isoxanthopterin--a fluorescent analogue of the nucleic acid base guanine.

Fluorescent nucleic acid base analogues are important spectroscopic tools for understanding local structure and dynamics of DNA and RNA. We studied the orientations and magnitudes of the electric dipole transition moments (EDTMs) of 6-methyl isoxanthopterin (6-MI), a fluorescent analogue of guanine that has been particularly useful in biological studies. Using a combination of absorption spectroscopy, linear dichroism (LD) and quantum chemical calculations, we identified six electronic transitions that occur within the 25,000-50,000 cm(-1) spectral range. Our results indicate that the two experimentally observed lowest-energy transitions, which occur at 29,687 cm(-1) (337 nm) and 34,596 cm(-1) (289 nm), are each polarized within the plane of the 6-MI base. A third in-plane polarized transition is experimentally observed at 47,547 cm(-1) (210 nm). The theoretically predicted orientation of the lowest-energy transition moment agrees well with experiment. Based on these results, we constructed an exciton model to describe the absorption spectra of a 6-MI dinucleotide-substituted double-stranded DNA construct. This model is in good agreement with the experimental data. The orientations and intensities of the low-energy electronic transitions of 6-MI reported here should be useful for studying local conformations of DNA and RNA in biologically important complexes.

Conformation and electronic population transfer in membrane-supported self-assembled porphyrin dimers by 2D fluorescence spectroscopy.

Two-dimensional fluorescence spectroscopy (2D FS) is applied to determine the conformation and femtosecond electronic population transfer in a dimer of magnesium meso tetraphenylporphyrin. The dimers are prepared by self-assembly of the monomer within the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. A theoretical framework to describe 2D FS experiments is presented, and a direct comparison is made between the observables of this measurement and those of 2D electronic spectroscopy (2D ES). The sensitivity of the method to varying dimer conformation is explored. A global multivariable fitting analysis of linear and 2D FS data indicates that the dimer adopts a "bent T-shaped" conformation. Moreover, the manifold of singly excited excitons undergoes rapid electronic dephasing and downhill population transfer on the time scale of ∼95 fs. The open conformation of the dimer suggests that its self-assembly is favored by an increase in entropy of the local membrane environment.

Compressed Sensing for Multidimensional Spectroscopy Experiments.

Compressed sensing is a processing method that significantly reduces the number of measurements needed to accurately resolve signals in many fields of science and engineering. We develop a two-dimensional variant of compressed sensing for multidimensional spectroscopy and apply it to experimental data. For the model system of atomic rubidium vapor, we find that compressed sensing provides an order-of-magnitude (about 10-fold) improvement in spectral resolution along each dimension, as compared to a conventional discrete Fourier transform, using the same data set. More attractive is that compressed sensing allows for random undersampling of the experimental data, down to less than 5% of the experimental data set, with essentially no loss in spectral resolution. We believe that by combining powerful resolution with ease of use, compressed sensing can be a powerful tool for the analysis and interpretation of ultrafast spectroscopy data.

Conformation of self-assembled porphyrin dimers in liposome vesicles by phase-modulation 2D fluorescence spectroscopy.

By applying a phase-modulation fluorescence approach to 2D electronic spectroscopy, we studied the conformation-dependent exciton coupling of a porphyrin dimer embedded in a phospholipid bilayer membrane. Our measurements specify the relative angle and separation between interacting electronic transition dipole moments and thus provide a detailed characterization of dimer conformation. Phase-modulation 2D fluorescence spectroscopy (PM-2D FS) produces 2D spectra with distinct optical features, similar to those obtained using 2D photon-echo spectroscopy. Specifically, we studied magnesium meso tetraphenylporphyrin dimers, which form in the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. Comparison between experimental and simulated spectra show that although a wide range of dimer conformations can be inferred by either the linear absorption spectrum or the 2D spectrum alone, consideration of both types of spectra constrain the possible structures to a "T-shaped" geometry. These experiments establish the PM-2D FS method as an effective approach to elucidate chromophore dimer conformation.