Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α‐amino acids were condensed into polypeptides before the emergence of enzymes remains ...unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α‐hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α‐hydroxy acids and α‐amino acids form depsipeptides—oligomers with a combination of ester and amide linkages—in model prebiotic reactions that are driven by wet–cool/dry–hot cycles. Through a combination of ester–amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long‐standing hypothesis that peptides might have arisen from ester‐based precursors.
Amino acids form peptide bonds when subjected to day–night cycles (wet–dry cycles) in the presence of hydroxy acids. Such a reaction could have occurred on the prebiotic Earth.
Both primary and secondary nucleation rates are considered in a model developed for unseeded batch crystallization. A carefully designed strategy was employed to minimize the effects of the ...stochastic nature of induction time; nucleation was induced at designed supersaturations on known temperature plateaus. Crystallization kinetics of paracetamol from ethanolic solutions were extracted from measurements of in situ solute concentrations and combined with sieve (ex situ) data on the final product. Parameters in models for primary and secondary nucleation and for crystal growth rate were estimated by fitting a full population balance model to the measurements, and the evolution of the crystal size distribution was compared against in situ estimation from focused-beam reflectance measurements using the technique that we previously developed. The resulting models suggest that primary nucleation produces fewer surviving crystals than had been expected and that most of the product crystals from the process involving a temperature plateau result from secondary nucleation.
The non-enzymatic cleavage rates of amide bonds located in peptides in aqueous solution is pH-dependent and involves two distinct mechanisms: direct hydrolysis (herein termed "scission") and ...intramolecular aminolysis by the N-terminal amine (herein termed "backbiting"). While amide bond cleavage has been previously characterized using a variety of peptides, no systematic study has yet been reported addressing the effect of the pH on the interplay between the two amide bond cleavage pathways. In this study, the cleavage rates of the glycine dimer (GG), the glycine trimer (GGG), and the cyclic dimer (cGG), as well as the alanine trimer (AAA), were measured at pH 3, 5, 7, and 10 at 95 °C employing quantification based on 1H NMR. The distinct rate constants for scission and backbiting processes were obtained by solving the differential rate equations associated with the proposed kinetic model. Generalizations concerning the relative importance of the various amide bond cleavage pathways at pH 3, 5, 7, and 10 are presented. In particular, scission dominates at pH 10, while backbiting dominates at neutral pH. At the acidic pH of 3, both backbiting and scission are significant. The model of the reaction network, used in this work, enables the quantification of these multiple competing mechanisms and can be applied to longer peptides and to similar types of reaction networks.
Defining pathways for amyloid assembly could impact therapeutic strategies for as many as 50 disease states. Here we show that amyloid assembly is subject to different forces regulating nucleation ...and propagation steps and provide evidence that the more global β-sheet/β-sheet facial complementarity is a critical determinant for amyloid nucleation and structural selection.
Nanofibers are a ubiquitous structural motif in a variety of functional materials. In the field of organic electronics, π–π-stacking of conjugated polymers leads to fibrillar morphologies with a wide ...array of fiber packing behavior. Fiber orientation and alignment are known to influence the charge transport properties of devices such as organic field effect transistors. The solution processing methods used to create these devices give rise to large variations in these structural parametershowever, they are only observable with imaging techniques such as atomic force microscopy (AFM). To bring more rigorous quantification of orientation and alignment to these materials, a comprehensive image analysis tool is introduced to quantify the two-dimensional orientation and alignment of nanofibers from AFM images. It has been developed in MATLAB and packaged as a stand-alone application, so that researchers with no computational expertise can produce publication-ready figures directly from their images. AFM frequently yields images with low contrast and moderate noise, making quantitative feature extraction a significant challenge. In this protocol, each image is analyzed in the context of an Orientation Map, in which nanofibers are thinned to single-pixel width and an orientation is extracted for each of these pixels. The Orientation Map is obtained through a five-step process: fiber smoothing by anisotropic diffusion filtering, contrast enhancement by top hat filtering, binarization by adaptive thresholding, skeletonization, and recovery of orientations from the result of diffusion filtering. Each step involves parameters that can be set using physical heuristics, which are examined in detail. This Orientation Map yields an orientation distribution and a plot of S 2D, an orientational order parameter, as a function of frame size. The image analysis procedure is used to quantify differences in P3HT nanofiber morphology induced by various solution processing recipes, as well as the effect of spin-coating when used to deposit solutions of nanofibers. All examples presented in this protocol can be reproduced from beginning to end using the included software, with visualizations produced at each stage of processing.
Perfectly ordered states are targets in diverse molecular to microscale systems involving, for example, atomic clusters, protein folding, protein crystallization, nanoparticle superlattices, and ...colloidal crystals. However, there is no obvious approach to control the assembly of perfectly ordered global free energy minimum structures; near-equilibrium assembly is impractically slow, and faster out-of-equilibrium processes generally terminate in defective states. Here, we demonstrate the rapid and robust assembly of perfect crystals by navigating kinetic bottlenecks using closed-loop control of electric field mediated crystallization of colloidal particles. An optimal policy is computed with dynamic programming using a reaction coordinate based dynamic model. By tracking real-time stochastic particle configurations and adjusting applied fields via feedback, the evolution of unassembled particles is guided through polycrystalline states into single domain crystals. This approach to controlling the assembly of a target structure is based on general principles that make it applicable to a broad range of processes from nano- to microscales (where tuning a global thermodynamic variable yields temporal control over thermal sampling of different states via their relative free energies).
There is great interest in DNA nanotechnology, but its use has been limited to aqueous or substantially hydrated media. The first assembly of a DNA nanostructure in a water‐free solvent, namely a ...low‐volatility biocompatible deep‐eutectic solvent composed of a 4:1 mixture of glycerol and choline chloride (glycholine), is now described. Glycholine allows for the folding of a two‐dimensional DNA origami at 20 °C in six days, whereas in hydrated glycholine, folding is accelerated (≤3 h). Moreover, a three‐dimensional DNA origami and a DNA tail system can be folded in hydrated glycholine under isothermal conditions. Glycholine apparently reduces the kinetic traps encountered during folding in aqueous solvent. Furthermore, folded structures can be transferred between aqueous solvent and glycholine. It is anticipated that glycholine and similar solvents will allow for the creation of functional DNA structures of greater complexity by providing a milieu with tunable properties that can be optimized for a range of applications and nanostructures.
Who needs water? DNA structures, such as a two‐dimensional DNA origami, can be folded in an anhydrous deep‐eutectic solvent composed of glycerol and choline chloride (“glycholine”). In hydrated glycholine, folding is accelerated, and more complex nanostructures can be isothermally folded, demonstrating that DNA self‐assembly kinetics can be tuned by changing solvent viscosity.
The RNA World hypothesis posits that RNA was once responsible for genetic information storage and catalysis. However, a prebiotic mechanism has yet to be reported for the replication of duplex RNA ...that could have operated before the emergence of polymerase ribozymes. Previously, we showed that a viscous solvent enables information transfer from one strand of long RNA duplex templates, overcoming 'the strand inhibition problem'. Here, we demonstrate that the same approach allows simultaneous information transfer from both strands of long duplex templates. An additional challenge for the RNA World is that structured RNAs (like those with catalytic activity) function poorly as templates in model prebiotic RNA synthesis reactions, raising the question of how a single sequence could serve as both a catalyst and as a replication template. Here, we show that a viscous solvent also facilitates the transition of a newly synthesized hammerhead ribozyme sequence from its inactive, duplex state to its active, folded state. These results demonstrate how fluctuating environmental conditions can allow a ribozyme sequence to alternate between acting as a template for replication and functioning as a catalyst, and illustrate the potential for temporally changing environments to enable molecular processes necessary for the origin of life.
The multiphase nature of slurries can make them difficult to process and monitor in real time. For example, the nuclear waste slurries present at the Hanford site in Washington State are ...multicomponent, multiphase, and inhomogeneous. Current analytical techniques for analyzing radioactive waste at Hanford rely on laboratory results from an on-site analytical laboratory, which can delay processing speed and create exposure risks for workers. However, in-line probes can provide an alternative route to collect the necessary composition information. In the present work, Raman spectroscopy and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy are tested on simulants of nuclear waste slurries containing up to 23.2 wt % solids. We observe ATR-FTIR spectroscopy to be effective in measuring the solution phase of the studied slurry systems (3.52% mean percent error), while Raman spectroscopy provides information about the suspended solids in the slurry system (18.21% mean percent error). In-line measurement of multicomponent solids typical of nuclear waste processing has been previously unreported. The composition of both the solution and solid phases is vital in ensuring stable glass formulation and effective disposal of nuclear waste at Hanford. Raman and ATR-FTIR spectroscopies can provide a safer and faster alternative for acquiring compositional information on nuclear waste slurries.
Intermediate dynamic assemblies are increasingly seen as necessary for the initial desolvation and organization of biomaterials to achieve their final crystalline order. Here we present a general ...peptide assembly model for two-step nucleation. The model predicts the phase transitions and equilibria between different phases by employing a combination of the Flory–Huggins parameter, the particle growth constant, and the binding energy to assemblies. Monte Carlo simulations are used to demonstrate how the system evolves from pure solution phases to the final thermodynamic assembly phase via an intermediate metastable particle phase. The final state of the system is determined by the solubility of the particle and assembly phases, where the phase with the lower solubility accumulates. A rare three-phase equilibrium exists when the solubilities of the particles and assemblies are similar. Experimental support for this model is achieved with assembly of the amyloid peptide Ac-KLVFFAE-NH2 (Aβ(16–22)) in mixed acetonitrile/water systems. Increasing the acetonitrile concentration decreases the number of particles, increases the particle size, and accelerates the assembly rate, all consistent with acetonitrile increasing the Aβ(16–22) peptide’s solubility of particles but with little influence on the stability of the assemblies. Taken together, our model captures the transition from the metastable particle phase to the higher order peptide assembly through two-step nucleation.
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