The Nature of the Spark Is a Pivotal Element in the Design of a Miller-Urey Experiment.

Miller and Urey applied electric sparks to a reducive mixture of CH4, NH3, and water to obtain a complex organic mixture including biomolecules. In this study, we examined the impact of temperature, initial pressure, ammonia concentration, and the spark generator on the chemical profile of a Miller-Urey-type prebiotic broth. We analyzed the broth composition using Gas Chromatography combined with Mass Spectroscopy (GC/MS). The results point towards strong compositional changes with the nature of the spark. Ammonia exhibited catalytic properties even with non-nitrogen-containing compounds. A more elevated temperature led to a higher variety of substances. We conclude that to reproduce such a broth as well as possible, all the studied parameters need to be tightly controlled, the most difficult and important being spark generation.

Protocol to identify amino acids bound to tRNA by aminoacylation using mass spectrometry.

tRNA-bound amino acids often need to be identified, for instance, in cases where different amino acids compete for binding to the same tRNA. Here, we present a mass-spectrometry-based protocol to determine the amino acids bound to tRNA by aminoacylation. We detail how to perform the aminoacylation reaction, the preparation of the aminoacyl-tRNA for measurement, and the mass spectrometric analysis. We use arginine acylation as an example; however, this protocol can be applied to any other amino acid.

3D single cell migration driven by temporal correlation between oscillating force dipoles.

Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices, do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement, and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-driven force dipoles at both sides of the nucleus, that locally and periodically pinch the matrix. The existence of a phase shift between the two dipoles is required for directed cell motion which manifests itself as cycles with finite area in the dipole-quadrupole diagram, a formal equivalence to the Purcell cycle. We confirm this mechanism by triggering local dipolar contractions with a laser. This leads to directed motion. Our study reveals that these cells control their motility by synchronizing dipolar forces distributed at front and back. This result opens new strategies to externally control cell motion as well as for the design of micro-crawlers.

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane.

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA Reconstituted in an All E. coli Cell-Free Expression System.

Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.

Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of l-lysine by l-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational l-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinked.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems.

Cell-free transcription-translation systems are a versatile tool to study gene expression, enzymatic reactions and biochemical regulation mechanisms. Because cell-free transcription-translation systems are often derived from cell lysates, many different substances, among them amino acids, are present. However, experiments concerning the incorporation of non-canonical amino acids into proteins require a system with negligible amounts of canonical analogs. Here we propose a two-step method for the removal of residual free lysine in an all Escherichia coli-based cell-free expression system. The first step consists of the expression of a high-lysine dummy protein. The second step consists of direct removal via binding between lysine and DNA. The presented method is an efficient, fast and simple way to remove residual lysine without altering the system ability to perform gene expression.

Bead-based assay for spatiotemporal gene expression control in cell-free transcription-translation systems.

Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a bead-based method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.

A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Large Amplitude Oscillatory Shear Rheology of Living Fibroblasts: Path-Dependent Steady States.

Mechanical properties of biological cells play a role in cell locomotion, embryonic tissue formation, and tumor migration among many other processes. Cells exhibit a complex nonlinear response to mechanical cues that is not understood. Cells may stiffen as well as soften, depending on the exact type of stimulus. Here we apply large-amplitude oscillatory shear to a monolayer of separated fibroblast cells suspended between two plates. Although we apply identical steady-state excitations, in response we observe different typical regimes that exhibit cell softening or cell stiffening to varying degrees. This degeneracy of the cell response can be linked to the initial paths that the instrument takes to go from cell rest to steady state. A model of cross-linked, force-bearing filaments submitted to steady-state excitation renders the different observed regimes with minor changes in parameters if the filaments are permitted to self-organize and form different spatially organized structures. We suggest that rather than a complex viscoelastic or plastic response, the different observed regimes reflect the emergence of different steady-state cytoskeletal conformations. A high sensitivity of the cytoskeletal rheology and structure to minor changes in parameters or initial conditions enables a cell to respond to mechanical requirements quickly and in various ways with only minor biochemical intervention. Probing path-dependent rheological changes constitutes a possibly very sensitive assessment of the cell cytoskeleton as a possible tool for medical diagnosis. Our observations show that the memory of subtle differences in earlier deformation paths must be taken into account when deciphering the cell mechanical response to large-amplitude deformations.

Hamming Distance as a Concept in DNA Molecular Recognition.

DNA microarrays constitute an in vitro example system of a highly crowded molecular recognition environment. Although they are widely applied in many biological applications, some of the basic mechanisms of the hybridization processes of DNA remain poorly understood. On a microarray, cross-hybridization arises from similarities of sequences that may introduce errors during the transmission of information. Experimentally, we determine an appropriate distance, called minimum Hamming distance, in which the sequences of a set differ. By applying an algorithm based on a graph-theoretical method, we find large orthogonal sets of sequences that are sufficiently different not to exhibit any cross-hybridization. To create such a set, we first derive an analytical solution for the number of sequences that include at least four guanines in a row for a given sequence length and eliminate them from the list of candidate sequences. We experimentally confirm the orthogonality of the largest possible set with a size of 23 for the length of 7. We anticipate our work to be a starting point toward the study of signal propagation in highly competitive environments, besides its obvious application in DNA high throughput experiments.

Chemical Analysis of a "Miller-Type" Complex Prebiotic Broth : Part II: Gas, Oil, Water and the Oil/Water-Interface.

We have analyzed the chemical variety obtained by Miller-Urey-type experiments using nuclear magnetic resonance (NMR) spectroscopy and coherent anti-Stokes Raman scattering (CARS) spectroscopy, gas chromatography followed by mass spectrometry (GC/MS) and two-dimensional gas chromatography followed by mass spectrometry (GCxGC/MS). In the course of a running Miller-Urey-type experiment, a hydrophobic organic layer emerged besides the hydrophilic aqueous phase and the gaseous phase that were initially present. The gas phase mainly consisted of aromatic compounds and molecules containing C≡C or C≡N triple bonds. The hydrophilic phase contained at least a few thousands of different molecules, primarily distributed in a range of 50 and 500 Da. The hydrophobic phase is characterized by carbon-rich, oil-like compounds and their amphiphilic derivatives containing oxygen with tensioactive properties. The presence of a wide range of oxidized molecules hints to the availability of oxygen radicals. We suggest that they intervene in the formation of alkylated polyethylene glycol (PEG) in the oil/water interface. CARS spectroscopy revealed distinct vibrational molecular signatures. In particular, characteristic spectral bands for cyanide compounds were observed if the broth was prepared with electric discharges in the gaseous phase. The characteristic spectral bands were absent if discharges were released onto the water surface. NMR spectroscopy on the same set of samples independently confirmed the observation. In addition, NMR spectroscopy revealed overall high chemical variability that suggests strong non-linearities due to interdependent, sequential reaction steps.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Tracking of plus-ends reveals microtubule functional diversity in different cell types.

Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics.

Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA) technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

Chemical Analysis of a "Miller-Type" Complex Prebiotic Broth: Part I: Chemical Diversity, Oxygen and Nitrogen Based Polymers.

In a famous experiment Stanley Miller showed that a large number of organic substances can emerge from sparking a mixture of methane, ammonia and hydrogen in the presence of water (Miller, Science 117:528-529, 1953). Among these substances Miller identified different amino acids, and he concluded that prebiotic events may well have produced many of Life's molecular building blocks. There have been many variants of the original experiment since, including different gas mixtures (Miller, J Am Chem Soc 77:2351-2361, 1955; Oró Nature 197:862-867, 1963; Schlesinger and Miller, J Mol Evol 19:376-382, 1983; Miyakawa et al., Proc Natl Acad Sci 99:14,628-14,631, 2002). Recently some of Miller's remaining original samples were analyzed with modern equipment (Johnson et al. Science 322:404-404, 2008; Parker et al. Proc Natl Acad Sci 108:5526-5531, 2011) and a total of 23 racemic amino acids were identified. To give an overview of the chemical variety of a possible prebiotic broth, here we analyze a "Miller type" experiment using state of the art mass spectrometry and NMR spectroscopy. We identify substances of a wide range of saturation, which can be hydrophilic, hydrophobic or amphiphilic in nature. Often the molecules contain heteroatoms, with amines and amides being prominent classes of molecule. In some samples we detect ethylene glycol based polymers. Their formation in water requires the presence of a catalyst. Contrary to expectations, we cannot identify any preferred reaction product. The capacity to spontaneously produce this extremely high degree of molecular variety in a very simple experiment is a remarkable feature of organic chemistry and possibly prerequisite for Life to emerge. It remains a future task to uncover how dedicated, organized chemical reaction pathways may have arisen from this degree of complexity.

Using cell monolayer rheology to probe average single cell mechanical properties.

The cell monolayer rheology technique consists of a commercial rotational rheometer that probes the mechanical properties of a monolayer of isolated cells. So far we have described properties of an entire monolayer. In this short communication, we show that we can deduce average single cell properties. Results are in very good agreement with earlier work on single cell mechanics. Our approach provides a mean of 105-106 adherent cells within a single experiment. This makes the results very reproducible. We extend our work on cell adhesion strength and deduce cell adhesion forces of fibroblast cells on fibronectin coated glass substrates.

Cell-free expression with the toxic amino acid canavanine.

Canavanine is a naturally occurring noncanonical amino acid, which is analogous to arginine. It is a potent antimetabolite and natural allelochemic agent, capable of affecting or blocking regulatory and catalytic reactions that involve arginine. Incorporated into proteins at arginine positions, canavanine can be detrimental to protein stability and functional integrity. Although incorporation of canavanine into proteins has long been documented, due to its toxicity, expression in Escherichia coli and other common hosts remains a considerable challenge. Here, we present a simple, cell-free expression system with markedly improved performance compared to heterologous expression. The cell-free expression system does not require any tuning besides substitution of arginine by canavanine. We show that our technique enables highly efficient protein expression in small volumes with arginine being fully replaced by canavanine for functional and structural studies.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Putative cholesterol-binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5.

Using molecular docking, we identified a cholesterol-binding site in the groove between transmembrane helices 1 and 7 near the inner membrane-water interface of the G protein-coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol-binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol-binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol-binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins.