Early Aggregation of Amyloid-ß(1-42) Studied by Fluorescence Correlation Spectroscopy.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting cognitive and memory abilities and is believed to be linked to the formation and accumulation of neurotoxic aggregates of the Amyloid-ß peptide (Aß). In particular, it is the formation of soluble pre-fibrillar oligomers within the early stage of Aß aggregation which is thought to represent a key step in the development of AD, thus underlining the interest in characterizing the aggregation process and the nature of these aggregates. In this context, fluorescence correlation spectroscopy (FCS) has emerged as a valuable alternative for the study of these systems in solution. Indeed, the use of FCS to study terminally labelled Aß provides a means to detect changes in the size and concentration of initially monomeric Aß samples by monitoring these fluorescently labelled species freely diffusing in solution with single-molecule resolution. Herein, we show how to employ FCS to study the early aggregation process of Aß(1-42) and how this can be used to estimate the critical concentration for oligomer formation and to characterize the aggregates formed.

Deactivation of a dimeric DNA-binding peptide through a palladium-mediated self-immolative cleavage.

Herein, we describe an approach for the on-demand disassembly of dimeric peptides using a palladium-mediated cleavage of a designed self-immolative linker. The utility of the strategy is demonstrated for the case of dimeric basic regions of bZIP transcription factors. While the dimer binds designed DNA sequences with good affinities, the peptide-DNA complex can be readily dismounted by addition of palladium reagents that trigger the cleavage of the spacer, and the release of unfunctional monomeric peptides.

Spectroscopic Characterization of Mitochondrial G-Quadruplexes.

Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.

Monitoring RNA dynamics in native transcriptional complexes.

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

Bioorthogonal Azide-Thioalkyne Cycloaddition Catalyzed by Photoactivatable Ruthenium(II) Complexes.

Tailored ruthenium sandwich complexes bearing photoresponsive arene ligands can efficiently promote azide-thioalkyne cycloaddition (RuAtAC) when irradiated with UV light. The reactions can be performed in a bioorthogonal manner in aqueous mixtures containing biological components. The strategy can also be applied for the selective modification of biopolymers, such as DNA or peptides. Importantly, this ruthenium-based technology and the standard copper-catalyzed azide-alkyne cycloaddition (CuAAC) proved to be compatible and mutually orthogonal.

A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition.

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.

Asymmetric base-pair opening drives helicase unwinding dynamics.

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.

Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes.

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

Biophysical Approaches to Bacterial Gene Regulation by Riboswitches.

The last decade has witnessed the discovery of a variety of non-coding RNA sequences that perform a broad range of crucial biological functions. Among these, the ability of certain RNA sequences, so-called riboswitches, has attracted considerable interest. Riboswitches control gene expression in response to the concentration of particular metabolites to which they bind without the need for any protein. These RNA switches not only need to adopt a very specific tridimensional structure to perform their function, but also their sequence has been evolutionary optimized to recognize a particular metabolite with high affinity and selectivity. Thus, riboswitches offer a unique opportunity to get fundamental insights into RNA plasticity and how folding dynamics and ligand recognition mechanisms have been efficiently merged to control gene regulation. Because riboswitch sequences have been mostly found in bacterial organisms controlling the expression of genes associated to the synthesis, degradation or transport of crucial metabolites for bacterial survival, they offer exciting new routes for antibiotic development in an era where bacterial resistance is more than ever challenging conventional drug discovery strategies. Here, we give an overview of the architecture, diversity and regulatory mechanisms employed by riboswitches with particular emphasis on the biophysical methods currently available to characterise their structure and functional dynamics.

Morphology-Specific Inhibition of ß-Amyloid Aggregates by 17ß-Hydroxysteroid Dehydrogenase Type 10.

A major hallmark of Alzheimer's disease (AD) is the formation of toxic aggregates of the ß-amyloid peptide (Aß). Given that Aß peptides are known to localise within mitochondria and interact with 17ß-HSD10, a mitochondrial protein expressed at high levels in AD brains, we investigated the inhibitory potential of 17ß-HSD10 against Aß aggregation under a range of physiological conditions. Fluorescence self-quenching (FSQ) of Aß(1-42) labelled with HiLyte Fluor 555 was used to evaluate the inhibitory effect under conditions established to grow distinct Aß morphologies. 17ß-HSD10 preferentially inhibits the formation of globular and fibrillar-like structures but has no effect on the growth of amorphous plaque-like aggregates at endosomal pH 6. This work provides insights into the dependence of the Aß-17ß-HSD10 interaction with the morphology of Aß aggregates and how this impacts enzymatic function.