Nonequilibrium Scattering/Evaporation Dynamics at the Gas-Liquid Interface: Wetted Wheels, Self-Assembled Monolayers, and Liquid Microjets.

ConspectusWe often teach or are taught in our freshman courses that there are three phases of matter─gas, liquid and solid─where the ordering reflects increasing complexity and strength of interaction between the molecular constituents. But arguably there is also a fascinating additional "phase" of matter associated with the microscopically thin interface (<10 molecules thick) between the gas and liquid, which is still poorly understood and yet plays a crucial role in fields ranging from chemistry of the marine boundary layer and atmospheric chemistry of aerosols to the passage of O2 and CO2 through alveolar sacs in our lungs. The work in this Account provides insights into three challenging new directions for the field, each embracing a rovibronically quantum-state-resolved perspective. Specifically, we exploit the powerful tools of chemical physics and laser spectroscopy to pose two fundamental questions. (i) At the microscopic level, do molecules in all internal quantum-states (e.g., vibrational, rotational, electronic) colliding with the interface "stick" with unit probability? (ii) Can reactive, scattering, and/or evaporating molecules at the gas-liquid interface avoid collisions with other species and thereby be observed in a truly "nascent" collision-free distribution of internal degrees of freedom? To help address these questions, we present studies in three different areas: (i) reactive scattering dynamics of F atoms with wetted-wheel gas-liquid interfaces, (ii) inelastic scattering of HCl from self-assembled monolayers (SAMs) via resonance-enhanced photoionization (REMPI)/velocity map imaging (VMI) methods, and (iii) quantum-state-resolved evaporation dynamics of NO at the gas-water interface. As a recurring theme, we find that molecular projectiles reactively, inelastically, or evaporatively scatter from the gas-liquid interface into internal quantum-state distributions substantially out of equilibrium with respect to the bulk liquid temperatures (TS). By detailed balance considerations, the data unambiguously indicate that even simple molecules exhibit rovibronic state dependences to how they "stick" to and eventually solvate into the gas-liquid interface. Such results serve to underscore the importance of quantum mechanics and nonequilibrium thermodynamics in energy transfer and chemical reactions at the gas-liquid interface. This nonequilibrium behavior may well make this rapidly emergent field of chemical dynamics at gas-liquid interfaces more complicated but even more interesting targets for further experimental/theoretical exploration.

Neurophysiological consequences of synapse loss in progressive supranuclear palsy.

Synaptic loss occurs early in many neurodegenerative diseases and contributes to cognitive impairment even in the absence of gross atrophy. Currently, for human disease there are few formal models to explain how cortical networks underlying cognition are affected by synaptic loss. We advocate that biophysical models of neurophysiology offer both a bridge from preclinical to clinical models of pathology and quantitative assays for experimental medicine. Such biophysical models can also disclose hidden neuronal dynamics generating neurophysiological observations such as EEG and magnetoencephalography. Here, we augment a biophysically informed mesoscale model of human cortical function by inclusion of synaptic density estimates as captured by 11C-UCB-J PET, and provide insights into how regional synapse loss affects neurophysiology. We use the primary tauopathy of progressive supranuclear palsy (Richardson's syndrome) as an exemplar condition, with high clinicopathological correlations. Progressive supranuclear palsy causes a marked change in cortical neurophysiology in the presence of mild cortical atrophy and is associated with a decline in cognitive functions associated with the frontal lobe. Using parametric empirical Bayesian inversion of a conductance-based canonical microcircuit model of magnetoencephalography data, we show that the inclusion of regional synaptic density-as a subject-specific prior on laminar-specific neuronal populations-markedly increases model evidence. Specifically, model comparison suggests that a reduction in synaptic density in inferior frontal cortex affects superficial and granular layer glutamatergic excitation. This predicted individual differences in behaviour, demonstrating the link between synaptic loss, neurophysiology and cognitive deficits. The method we demonstrate is not restricted to progressive supranuclear palsy or the effects of synaptic loss: such pathology-enriched dynamic causal models can be used to assess the mechanisms of other neurological disorders, with diverse non-invasive measures of pathology, and is suitable to test the effects of experimental pharmacology.

High-resolution infrared spectroscopy of jet cooled cyclobutyl in the α-CH stretch region: large-amplitude puckering dynamics in a 4-membered ring radical.

Gas-phase cyclobutyl radical (c-C4H7) is generated at a rotational temperature of Trot = 26(1) K in a slit-jet discharge mixture of 70% Ne/30% He and 0.5-0.6% cyclobromobutane (c-C4H7Br). A fully rovibrationally resolved absorption spectrum of the α-CH stretch fundamental band between 3062.9 cm-1 to 3075.7 cm-1 is obtained and analyzed, yielding first precision structural and dynamical information for this novel radical species. The α-CH stretch band origin is determined to be 3068.7887(4) cm-1, which implies only a modest (≈0.8 cm-1) blue shift from rotationally unresolved infrared spectroscopic studies of cyclobutyl radicals in liquid He droplets [A. R. Brown, P. R. Franke and G. E. Douberly, J. Phys. Chem. A, 2017, 121, 7576-7587]. Of particular dynamical interest, a one-dimensional potential energy surface with respect to the ring puckering coordinate is computed at CCSD(T)/ANO2 level of theory and reveals a double minimum Cs puckered geometry, separated by an exceedingly shallow planar C2v transition state barrier (Ebarr ≈ 1 cm-1). Numerical solutions on this double minimum potential yield a zero-point energy for the ground state (Ezero-point ≈ 27 cm-1) greatly in excess of the interconversion barrier. This is indicative of highly delocalized, large amplitude motion of the four-membered ring structure, for which proper vibrationally averaging of the moment of inertia tensor reproduces the experimentally determined inertial defect remarkably well. Finally, intensity alternation in the experimental spectrum due to nuclear spin statistics upon exchange of three indistinguishable H atom pairs (IH = ½) matches Ka + Kc = even : odd = 36 : 28 predictions, implying that the unpaired electron in the radical center lies in an out-of-plane pπ orbital. Thus, high-resolution infrared spectroscopy provides first experimental confirmation of a shallow double minimum ring puckering potential with a highly delocalized ground state wave function peaked at a planar C2v transition state geometry consistent with a cyclobutyl π radical.

Ultrasensitive multispecies spectroscopic breath analysis for real-time health monitoring and diagnostics.

Breath analysis enables rapid, noninvasive diagnostics, as well as long-term monitoring of human health, through the identification and quantification of exhaled biomarkers. Here, we demonstrate the remarkable capabilities of mid-infrared (mid-IR) cavity-enhanced direct-frequency comb spectroscopy (CE-DFCS) applied to breath analysis. We simultaneously detect and monitor as a function of time four breath biomarkers-[Formula: see text]OH, [Formula: see text], [Formula: see text]O, and HDO-as well as illustrate the feasibility of detecting at least six more ([Formula: see text]CO, [Formula: see text], OCS, [Formula: see text], [Formula: see text], and [Formula: see text]) without modifications to the experimental apparatus. We achieve ultrahigh detection sensitivity at the parts-per-trillion level. This is made possible by the combination of the broadband spectral coverage of a frequency comb, the high spectral resolution afforded by the individual comb teeth, and the sensitivity enhancement resulting from a high-finesse cavity. Exploiting recent advances in frequency comb, optical coating, and photodetector technologies, we can access a large variety of biomarkers with strong carbon-hydrogen-bond spectral signatures in the mid-IR.

Neurophysiological and Brain Structural Markers of Cognitive Frailty Differ from Alzheimer's Disease.

With increasing life span and prevalence of dementia, it is important to understand the mechanisms of cognitive aging. Here, we focus on a subgroup of the population we term "cognitively frail," defined by reduced cognitive function in the absence of subjective memory complaints, or a clinical diagnosis of dementia. Cognitive frailty is distinct from cognitive impairment caused by physical frailty. It has been proposed to be a precursor to Alzheimer's disease, but may alternatively represent one end of a nonpathologic spectrum of cognitive aging. We test these hypotheses in humans of both sexes, by comparing the structural and neurophysiological properties of a community-based cohort of cognitive frail adults, to people presenting clinically with diagnoses of Alzheimer's disease or mild cognitive impairment, and community-based cognitively typical older adults. Cognitive performance of the cognitively frail was similar to those with mild cognitive impairment. We used a novel cross-modal paired-associates task that presented images followed by sounds, to induce physiological responses of novelty and associative mismatch, recorded by EEG/MEG. Both controls and cognitively frail showed stronger mismatch responses and larger temporal gray matter volume, compared with people with mild cognitive impairment and Alzheimer's disease. Our results suggest that community-based cognitively frail represents a spectrum of normal aging rather than incipient Alzheimer's disease, despite similar cognitive function. Lower lifelong cognitive reserve, hearing impairment, and cardiovascular comorbidities might contribute to the etiology of the cognitive frailty. Critically, community-based cohorts of older adults with low cognitive performance should not be interpreted as representing undiagnosed Alzheimer's disease.SIGNIFICANCE STATEMENT The current study investigates the neural signatures of cognitive frailty in relation to healthy aging and Alzheimer's disease. We focus on the cognitive aspect of frailty and show that, despite performing similarly to the patients with mild cognitive impairment, a cohort of community-based adults with poor cognitive performance do not show structural atrophy or neurophysiological signatures of Alzheimer's disease. Our results call for caution before assuming that cognitive frailty represents latent Alzheimer's disease. Instead, the cognitive underperformance of cognitively frail adults could result in cumulative effects of multiple psychosocial risk factors over the lifespan, and medical comorbidities.

Causal Evidence for the Multiple Demand Network in Change Detection: Auditory Mismatch Magnetoencephalography across Focal Neurodegenerative Diseases.

The multiple demand (MD) system is a network of fronto-parietal brain regions active during the organization and control of diverse cognitive operations. It has been argued that this activation may be a nonspecific signal of task difficulty. However, here we provide convergent evidence for a causal role for the MD network in the "simple task" of automatic auditory change detection, through the impairment of top-down control mechanisms. We employ independent structure-function mapping, dynamic causal modeling (DCM), and frequency-resolved functional connectivity analyses of MRI and magnetoencephalography (MEG) from 75 mixed-sex human patients across four neurodegenerative syndromes [behavioral variant fronto-temporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), posterior cortical atrophy (PCA), and Alzheimer's disease mild cognitive impairment with positive amyloid imaging (ADMCI)] and 48 age-matched controls. We show that atrophy of any MD node is sufficient to impair auditory neurophysiological response to change in frequency, location, intensity, continuity, or duration. There was no similar association with atrophy of the cingulo-opercular, salience or language networks, or with global atrophy. MD regions displayed increased functional but decreased effective connectivity as a function of neurodegeneration, suggesting partially effective compensation. Overall, we show that damage to any of the nodes of the MD network is sufficient to impair top-down control of sensation, providing a common mechanism for impaired change detection across dementia syndromes.SIGNIFICANCE STATEMENT Previous evidence for fronto-parietal networks controlling perception is largely associative and may be confounded by task difficulty. Here, we use a preattentive measure of automatic auditory change detection [mismatch negativity (MMN) magnetoencephalography (MEG)] to show that neurodegeneration in any frontal or parietal multiple demand (MD) node impairs primary auditory cortex (A1) neurophysiological response to change through top-down mechanisms. This explains why the impaired ability to respond to change is a core feature across dementias, and other conditions driven by brain network dysfunction, such as schizophrenia. It validates theoretical frameworks in which neurodegenerating networks upregulate connectivity as partially effective compensation. The significance extends beyond network science and dementia, in its construct validation of dynamic causal modeling (DCM), and human confirmation of frequency-resolved analyses of animal neurodegeneration models.

Non-equilibrium dynamics at the gas-liquid interface: State-resolved studies of NO evaporation from a benzyl alcohol liquid microjet.

First measurements of internal quantum-state distributions for nitric oxide (NO) evaporating from liquid benzyl alcohol are presented over a broad range of temperatures, performed by liquid-microjet techniques in an essentially collision-free regime, with rotational/spin-orbit populations in the 2Π1/2,3/2 manifolds measured by laser-induced fluorescence. The observed rotational distributions exhibit highly linear (i.e., thermal) Boltzmann plots but notably reflect rotational temperatures (Trot) as much as 30 K lower than the liquid temperature (Tjet). A comparable lack of equilibrium behavior is also noted in the electronic degrees of freedom but with populations corresponding to spin-orbit temperatures (TSO) consistently higher than Trot by ∼15 K. These results unambiguously demonstrate evaporation into a non-equilibrium distribution, which, by detailed-balance considerations, predict quantum-state-dependent sticking coefficients for incident collisions of NO at the gas-liquid interface. Comparison and parallels with previous experimental studies of NO thermal desorption and molecular-beam scattering in other systems are discussed, which suggests the emergence of a self-consistent picture for the non-equilibrium dynamics.

Regulating and Directionally Controlling Electron Emission from Gold Nanorods with Silica Coatings.

Dielectric coatings offer a versatile means of manipulating hot carrier emission from nanoplasmonic systems for emerging nanocatalysis and photocathode applications, with uniform coatings acting as regulators and nonuniform coatings providing directional photocurrent control. However, the mechanisms for electron emission through dense and mesoporous silica (SiO2) coatings require further examination. Here, we present a systematic investigation of photoemission from single gold nanorods as a function of dense versus mesoporous silica coating thicknesses. Studies with dense coatings on gold nanostructures clarify the short (∼1 nm) attenuation length responsible for severely reduced transmission through the silica conduction band. By contrast, mesoporous silica is much more transmissive, and a simple geometric model quantitatively recapitulates the electron escape probability through nanoscopic porous channels. Finally, photoelectron velocity map imaging (VMI) studies of nanorods with coating defects verify that photoemission occurs preferentially through the thinner regions, illustrating new opportunities for designing photocurrent distributions on the nanoscale.

A multi-site, multi-participant magnetoencephalography resting-state dataset to study dementia: The BioFIND dataset.

Early detection of Alzheimer's Disease (AD) is vital to reduce the burden of dementia and for developing effective treatments. Neuroimaging can detect early brain changes, such as hippocampal atrophy in Mild Cognitive Impairment (MCI), a prodromal state of AD. However, selecting the most informative imaging features by machine-learning requires many cases. While large publically-available datasets of people with dementia or prodromal disease exist for Magnetic Resonance Imaging (MRI), comparable datasets are missing for Magnetoencephalography (MEG). MEG offers advantages in its millisecond resolution, revealing physiological changes in brain oscillations or connectivity before structural changes are evident with MRI. We introduce a MEG dataset with 324 individuals: patients with MCI and healthy controls. Their brain activity was recorded while resting with eyes closed, using a 306-channel MEG scanner at one of two sites (Madrid or Cambridge), enabling tests of generalization across sites. A T1-weighted MRI is provided to assist source localisation. The MEG and MRI data are formatted according to international BIDS standards and analysed freely on the DPUK platform (https://portal.dementiasplatform.uk/Apply). Here, we describe this dataset in detail, report some example (benchmark) analyses, and consider its limitations and future directions.

GABAergic cortical network physiology in frontotemporal lobar degeneration.

The clinical syndromes caused by frontotemporal lobar degeneration are heterogeneous, including the behavioural variant frontotemporal dementia (bvFTD) and progressive supranuclear palsy. Although pathologically distinct, they share many behavioural, cognitive and physiological features, which may in part arise from common deficits of major neurotransmitters such as γ-aminobutyric acid (GABA). Here, we quantify the GABAergic impairment and its restoration with dynamic causal modelling of a double-blind placebo-controlled crossover pharmaco-magnetoencephalography study. We analysed 17 patients with bvFTD, 15 patients with progressive supranuclear palsy, and 20 healthy age- and gender-matched controls. In addition to neuropsychological assessment and structural MRI, participants undertook two magnetoencephalography sessions using a roving auditory oddball paradigm: once on placebo and once on 10 mg of the oral GABA reuptake inhibitor tiagabine. A subgroup underwent ultrahigh-field magnetic resonance spectroscopy measurement of GABA concentration, which was reduced among patients. We identified deficits in frontotemporal processing using conductance-based biophysical models of local and global neuronal networks. The clinical relevance of this physiological deficit is indicated by the correlation between top-down connectivity from frontal to temporal cortex and clinical measures of cognitive and behavioural change. A critical validation of the biophysical modelling approach was evidence from parametric empirical Bayes analysis that GABA levels in patients, measured by spectroscopy, were related to posterior estimates of patients' GABAergic synaptic connectivity. Further evidence for the role of GABA in frontotemporal lobar degeneration came from confirmation that the effects of tiagabine on local circuits depended not only on participant group, but also on individual baseline GABA levels. Specifically, the phasic inhibition of deep cortico-cortical pyramidal neurons following tiagabine, but not placebo, was a function of GABA concentration. The study provides proof-of-concept for the potential of dynamic causal modelling to elucidate mechanisms of human neurodegenerative disease, and explains the variation in response to candidate therapies among patients. The laminar- and neurotransmitter-specific features of the modelling framework, can be used to study other treatment approaches and disorders. In the context of frontotemporal lobar degeneration, we suggest that neurophysiological restoration in selected patients, by targeting neurotransmitter deficits, could be used to bridge between clinical and preclinical models of disease, and inform the personalized selection of drugs and stratification of patients for future clinical trials.

High-resolution infrared spectroscopy of supersonically cooled singlet carbenes: Bromomethylene (HCBr) in the CH stretch region.

First high-resolution spectra of cold (∼35 K) singlet bromomethylene HCBr in the CH stretching (v1) region from 2770 to 2850 cm-1 are reported using near quantum shot-noise limited laser absorption methods in a slit jet supersonic discharge expansion source. Three rovibrational bands are identified at high S/N (20:1-40:1) and rotationally assigned to (i) the CH stretch fundamental (v1) band X̃1,0,0←X̃0,0,0 and (ii) vibrational hot bands [X̃(1,1,0)←X̃(0,1,0) and X̃(1,0,1)←X̃(0,0,1)] arising from vibrationally excited HCBr populated in the discharge with single quanta in either the H-C-Br bend (v2) or C-Br stretch (v3) modes. Precision rotational constants are reported for a total of six states, with an experimentally determined CH stretch vibrational frequency (2799.38 cm-1) in good agreement with previous low-resolution fluorescence studies [M. Deselnicu et al., J. Chem. Phys. 124(13), 134302 (2006)]. Detailed analysis of the fundamental v1 band highlights the presence of perturbations in the X̃1,0,0 level, which we tentatively attribute to arise from the nearby triplet state ã(0,0,1) through spin-orbit interaction or the multiple quanta X̃0,2,1 singlet state via c-type Coriolis coupling. Reduced-Doppler resolution (60 MHz) in the slit-jet IR spectrometer permits for clear observation of a nuclear spin hyperfine structure, with experimental line shapes well reproduced by nuclear quadrupole/spin-rotation coupling constants from microwave studies [C. Duan et al., J. Mol. Spectrosc. 220(1), 113-121 (2003)]. Finally, the a-type to b-type transition intensity ratio for the fundamental CH stretch band is notably larger than that predicted by using a bond-dipole model, which from high level ab initio quantum calculations [CCSD(T)/PVQZ] can be attributed to vibrationally induced "charge-sloshing" of electron density along the polar C-Br bond.

Formation and detection of metastable formic acid in a supersonic expansion: High resolution infrared spectroscopy of the jet-cooled cis-HCOOH conformer.

High-resolution direct absorption infrared spectra of metastable cis-formic acid (HCOOH) trapped in a cis-well resonance behind a 15 kcal/mol barrier are reported for the first time, with the energetically unstable conformer produced in a supersonic slit plasma expansion of trans-formic acid/H2 mixtures. We present a detailed high-resolution rovibrational analysis for cis-formic acid species in the OH stretch (ν1) fundamental, providing first precision vibrational band origin, rotational constants, and term values, which in conjunction with ab initio calculations at the couple-cluster with single, double, and perturbative triple [CCSD(T)]/ANOn (n = 0, 1, 2) level support the experimental assignments and establish critical points on the potential energy surface for internal rotor trans-to-cis isomerization. Relative intensities for a- and b-type transitions observed in the spectra permit the transition dipole moment components to be determined in the body fixed frame and prove to be in good agreement with ab initio CCSD(T) theoretical estimates but in poor agreement with simple bond-dipole predictions. The observed signal dependence on H2 in the discharge suggests the presence of a novel H atom radical chemical mechanism for strongly endothermic "up-hill" internal rotor isomerization between trans- and cis-formic acid conformers.

High-resolution CH stretch spectroscopy of jet-cooled cyclopentyl radical: First insights into equilibrium structure, out-of-plane puckering, and IVR dynamics.

First, high-resolution sub-Doppler infrared spectroscopic results for cyclopentyl radical (C5H9) are reported on the α-CH stretch fundamental with suppression of spectral congestion achieved by adiabatic cooling to Trot ≈ 19(4) K in a slit jet expansion. Surprisingly, cyclopentyl radical exhibits a rotationally assignable infrared spectrum, despite 3N - 6 = 36 vibrational modes and an upper vibrational state density (ρ ≈ 40-90 #/cm-1) in the critical regime (ρ ≈ 100 #/cm-1) necessary for onset of intramolecular vibrational relaxation (IVR) dynamics. Such high-resolution data for cyclopentyl radical permit detailed fits to a rigid-rotor asymmetric top Hamiltonian, initial structural information for ground and vibrationally excited states, and opportunities for detailed comparison with theoretical predictions. Specifically, high level ab initio calculations at the coupled-cluster singles, doubles, and perturbative triples (CCSD(T))/ANO0, 1 level are used to calculate an out-of-plane bending potential, which reveals a C2 symmetry double minimum 1D energy surface over a C2v transition state. The inversion barrier [Vbarrier ≈ 3.7(1) kcal/mol] is much larger than the effective moment of inertia for out-of-plane bending, resulting in localization of the cyclopentyl wavefunction near its C2 symmetry equilibrium geometry and tunneling splittings for the ground state too small (<1 MHz) to be resolved under sub-Doppler slit jet conditions. The persistence of fully resolved high-resolution infrared spectroscopy for such large cyclic polyatomic radicals at high vibrational state densities suggests a "deceleration" of IVR for a cycloalkane ring topology, much as low frequency torsion/methyl rotation degrees of freedom have demonstrated a corresponding "acceleration" of IVR processes in linear hydrocarbons.

GABA-ergic Dynamics in Human Frontotemporal Networks Confirmed by Pharmaco-Magnetoencephalography.

To bridge the gap between preclinical cellular models of disease and in vivo imaging of human cognitive network dynamics, there is a pressing need for informative biophysical models. Here we assess dynamic causal models (DCM) of cortical network responses, as generative models of magnetoencephalographic observations during an auditory oddball roving paradigm in healthy adults. This paradigm induces robust perturbations that permeate frontotemporal networks, including an evoked 'mismatch negativity' response and transiently induced oscillations. Here, we probe GABAergic influences in the networks using double-blind placebo-controlled randomized-crossover administration of the GABA reuptake inhibitor, tiagabine (oral, 10 mg) in healthy older adults. We demonstrate the facility of conductance-based neural mass mean-field models, incorporating local synaptic connectivity, to investigate laminar-specific and GABAergic mechanisms of the auditory response. The neuronal model accurately recapitulated the observed magnetoencephalographic data. Using parametric empirical Bayes for optimal model inversion across both drug sessions, we identify the effect of tiagabine on GABAergic modulation of deep pyramidal and interneuronal cell populations. We found a transition of the main GABAergic drug effects from auditory cortex in standard trials to prefrontal cortex in deviant trials. The successful integration of pharmaco- magnetoencephalography with dynamic causal models of frontotemporal networks provides a potential platform on which to evaluate the effects of disease and pharmacological interventions.SIGNIFICANCE STATEMENT Understanding human brain function and developing new treatments require good models of brain function. We tested a detailed generative model of cortical microcircuits that accurately reproduced human magnetoencephalography, to quantify network dynamics and connectivity in frontotemporal cortex. This approach identified the effect of a test drug (GABA-reuptake inhibitor, tiagabine) on neuronal function (GABA-ergic dynamics), opening the way for psychopharmacological studies in health and disease with the mechanistic precision afforded by generative models of the brain.

Synthesis and applications of RNAs with position-selective labelling and mosaic composition.

Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.

Smaller molecules crowd better: Crowder size dependence revealed by single-molecule FRET studies and depletion force modeling analysis.

The cell is an extremely crowded environment, which is known to have a profound impact on the thermodynamics, functionality, and conformational stability of biomolecules. Speculations from recent theoretical molecular dynamics studies suggest an intriguing size dependence to such purely entropic crowding effects, whereby small molecular weight crowders under constant enthalpy conditions are more effective than larger crowders on a per volume basis. If experimentally confirmed, this would be profoundly significant, as the cellular cytoplasm is also quite concentrated in smaller molecular weight solutes such as inorganic ions, amino acids, and various metabolites. The challenge is to perform such studies isolating entropic effects under isoenthalpic conditions. In this work, we first present results from single-molecule FRET spectroscopy (smFRET) on the molecular size-dependent crowding stabilization of a simple RNA tertiary motif (the GAAA tetraloop-tetraloop receptor), indeed providing evidence in support of the surprising notion in the crowding literature that "smaller is better." Specifically, systematic smFRET studies as a function of crowder solute size reveal that smaller molecules both significantly increase the RNA tertiary folding rate and, yet, simultaneously decrease the unfolding rate, predicting strongly size-dependent stabilization of RNA tertiary structures under crowded cellular conditions. The size dependence of these effects has been explored via systematic variation of crowder size over a broad range of molecular weights (90-3000 amu). Furthermore, corresponding temperature dependent studies indicate the systematic changes in the folding equilibrium to be predominantly entropic in origin, i.e., consistent with a fundamental picture of entropic molecular crowding without additional enthalpic interactions. Most importantly, all trends in the single-molecule crowding data can be quantitatively recapitulated by a simple analytic depletion force model, whereby excluded volume interactions represent the major thermodynamic driving force toward folding. Our study, thus, not only provides experimental evidence and theoretical support for small molecule crowding but also predicts further enhancement of crowding effects for even smaller molecules on a per volume basis.

Single-molecule kinetic studies of DNA hybridization under extreme pressures.

Hydrostatic pressure can perturb biomolecular function by altering equilibrium structures and folding dynamics. Its influences are particularly important to deep sea organisms, as maximum pressures reach ≈1100 bar at the bottom of the ocean as a result of the rapid increase in hydraulic pressure (1 bar every 10 meters) under water. In this work, DNA hybridization kinetics has been studied at the single molecule level with external, tunable pressure control (Pmax≈ 1500 bar), realized by incorporating a mechanical hydraulic capillary sample cell into a confocal fluorescence microscope. We find that the DNA hairpin construct promotes unfolding ("denatures") with increasing pressure by simultaneously decelerating and accelerating the unimolecular rate constants for folding and unfolding, respectively. The single molecule kinetics is then investigated via pressure dependent van't Hoff analysis to infer changes in the thermodynamic molar volume, which unambiguously reveals that the effective DNA plus solvent volume increases (ΔV0 > 0) along the folding coordinate. Cation effects on the pressure dependent kinetics are also explored as a function of monovalent [Na+]. In addition to stabilizing the overall DNA secondary structure, sodium ions at low concentrations are also found to weaken any pressure dependence for the folding kinetics, but with these effects quickly saturating at physiologically relevant levels of [Na+]. In particular, the magnitudes of the activation volumes for the DNA dehybridization (ΔV) are significantly reduced with increasing [Na+], suggesting that sodium cations help DNA adopt a more fold-like transition state configuration.

High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation.

Deep sea biology is known to thrive at pressures up to ≈1 kbar, which motivates fundamental biophysical studies of biomolecules under such extreme environments. In this work, the conformational equilibrium of the lysine riboswitch has been systematically investigated by single molecule FRET (smFRET) microscopy at pressures up to 1500 bar. The lysine riboswitch preferentially unfolds with increasing pressure, which signals an increase in free volume (ΔV0 > 0) upon folding of the biopolymer. Indeed, the effective lysine binding constant increases quasi-exponentially with pressure rise, which implies a significant weakening of the riboswitch-ligand interaction in a high-pressure environment. The effects of monovalent/divalent cations and osmolytes on folding are also explored to acquire additional insights into cellular mechanisms for adapting to high pressures. For example, we find that although Mg2+ greatly stabilizes folding of the lysine riboswitch (ΔΔG0 < 0), there is negligible impact on changes in free volume (ΔΔV0 ≈ 0) and thus any pressure induced denaturation effects. Conversely, osmolytes (commonly at high concentrations in deep sea marine species) such as the trimethylamine N-oxide (TMAO) significantly reduce free volumes (ΔΔV0 < 0) and thereby diminish pressure-induced denaturation. We speculate that, besides stabilizing RNA structure, enhanced levels of TMAO in cells might increase the dynamic range for competent riboswitch folding by suppressing the pressure-induced denaturation response. This in turn could offer biological advantage for vertical migration of deep-sea species, with impacts on food searching in a resource limited environment.

Correction: High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation.

Correction for 'High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation' by Hsuan-Lei Sung et al., Phys. Chem. Chem. Phys., 2020, DOI: .

Continuous angular control over anisotropic photoemission from isotropic gold nanoshells.

A variety of applications rely on the efficient generation of hot carriers within metal nanoparticles and charge transfer to surrounding molecules or materials. The optimization of such processes requires a detailed understanding of excited carrier spatial, temporal, and momentum distributions, which also leads to opportunities for active optical control over hot carrier dynamics on nanometer and femtosecond scales. Such capabilities are emerging in nanoplasmonic systems and typically rely on tuning optical polarization and/or frequency to selectively excite one or more discrete hot spots defined by the particle geometry. Here, we introduce a unique case in which hot electron excitation and emission distributions can instead be continuously controlled via linear laser polarization in the azimuthal plane of a gold nanoshell supported on a substrate. In this configuration, it is the laser field that breaks the azimuthal symmetry of the supported nanoshell and determines the plasmonic field distribution. Using angle-resolved photoelectron velocity map imaging, we find that the hot electrons are predominantly emitted orthogonal to the nanoshell dipolar surface plasmon resonance axis defined by the laser polarization. Furthermore, such anisotropic emission is only observed for nanoshells, while solid gold nanospheres are found to be isotropic emitters. We show that all of these effects are recapitulated via simulation of the plasmonic electric field distributions within the nanoparticle volume and ballistic Monte Carlo modeling of the hot electron dynamics. These results demonstrate a highly predictive level of understanding of the underlying physics and possibilities for ultrafast spatiotemporal control over hot carrier dynamics.