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Microscale thermophoresis (MST) is a versatile technique to measure binding affinities of binder–ligand systems, based on the directional movement of molecules in a temperature gradient. We extended MST to measure binding kinetics as well as binding affinity in a single experiment by increasing the thermal dissipation of the sample. The kinetic relaxation fingerprints were derived from the fluorescence changes during thermodynamic re‐equilibration of the sample after local heating. Using this method, we measured DNA hybridization on‐rates and off‐rates in the range 104–106 m−1 s−1 and 10−4–10−1 s−1, respectively. We observed the expected exponential dependence of the DNA hybridization off‐rates on salt concentration, strand length and inverse temperature. The measured on‐rates showed a linear dependence on salt concentration and weak dependence on strand length and temperature. For biomolecular interactions with large enthalpic contributions, the kinetic MST technique offers a robust, cost‐effective and immobilization‐free determination of kinetic rates and binding affinity simultaneously, even in crowded solutions. [ABSTRACT FROM AUTHOR]

How life emerged is one of the major questions that remains to be answered. Apart from being of interest for completeness of biology, it is also a very interesting study case from the vantage point of physics. Living organisms are inherently non-equilibrium systems. Since non-equilibrium thermodynamics is still a developing field, the emergence of life is a highly interesting study case. Here we present the progress we have made during the last few years, employing experimental biophysics to capture the mechanisms that could eventually lead to the emergence of life. We show how a simple non-equilibrium system, a thermal gradient, gives rise to a range of relevant phenomena, in particular, polymerization, elongation, and replication of DNA molecules as well as demixing of mixed DNA sequences into sequence-pure hydrogels. 1 Introduction 2 Thermophoresis, the Biased Movement of Molecules in Thermal Gradients 3 The Dynamical Behavior of Accumulated Molecules 4 Overcoming Spiegelman’s Monster 5 Sequence Purification and Oscillations by a Gel-Phase Transition in Thermal Gradients 6 Loose Ends 7 Discussion and Conclusion [ABSTRACT FROM AUTHOR]

DNA phase transitions are often induced by the addition of condensation agents or by dry concentration. Herein, we show that the non-equilibrium setting of a moderate heat flow across a water-filled chamber separates and gelates DNA strands with single-base resolution. A dilute mix of DNA with two slightly different gel-forming sequences separates into sequence-pure hydrogels under constant physiological solvent conditions. A single base change in a 36 mer DNA inhibits gelation. Only sequences with the ability to form longer strands are concentrated, further elongated, and finally gelated by length-dependent thermal trapping. No condensation agents, such as multivalent ions, were added. Equilibrium aggregates from dry concentration did not show any sequence separation. RNA is expected to behave identically owing to its equal thermophoretic properties. The highly sequence-specific phase transition points towards new possibilities for non-equilibrium origins of life. [ABSTRACT FROM AUTHOR]

A combination of thermophoresis and fluid flow can be used to trap molecules and particles. We show that heating by scanning motion of an elongated laser spot creates a strong thermal trap. Additionally, it induces a global fluid flow that feeds the trap. Such "thermal sieve" can accumulate molecules from a large surrounding region within seconds into a 10 lm spot. Numerical modeling gives a quantitative prediction of the effect. Traps can be dynamically created, relocated, and tuned, which can be used for particle sorting. [ABSTRACT FROM AUTHOR]

The complexity of biology requires that measurements of biomolecular interactions be performed inside living cells. While electrophoresis inside cells is prohibited by the cell membrane, the movement of molecules along a temperature gradient appears feasible. This thermophoresis could be used to quantify binding affinities in vitro at picomolar levels and perform pharmaceutical fragment screens. Here we changed the measurement paradigm to enable measurements inside living cells. The temperature gradient is now applied along the optical axis and measures thermophoretic properties for each pixel of the camera image. We verify the approach for polystyrene beads and DNA of various lengths using finite element modeling. Thermophoresis inside living cells is able to record thermophoretic mobilities and intracellular diffusion coefficients across the whole cytoplasm. Interestingly, we find a 30-fold reduced diffusion coefficient inside the cell, indicating that molecular movement across the cell cytoplasm is slowed down due to molecular crowding. [ABSTRACT FROM AUTHOR]

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil-surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP-aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536-well plate format make the method high-throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label-free drug discovery screening. [ABSTRACT FROM AUTHOR]

Environmentally responsive polymers are becoming increasingly important in the biomaterials field for use as diagnostic reagents, drug carriers, and tissue engineering scaffolds. Characterizing polymer phase transitions by cloud point curves typically requires large milliliter volumes of sample at high micromolar solution concentrations. Here we present a method based on quantification of thermophoretic Soret diffusion that allows determination of polymer phase transitions using only ∼1 μL of liquid at dilute nanomolar concentrations, effectively reducing the amount of sample required by a factor of 106. We prepared an oligo(ethylene glycol) (OEG) methyl ether methacrylate copolymer via RAFT polymerization. End-group modification with fluorescent BODIPY-maleimide provided a dye-labeled pOEG-BODIPY conjugate with a lower critical solution temperature (LCST) in the range of ∼25-35 °C. Thermophoresis measurements in dilute solution demonstrated a marked change in polymer thermodiffusion in the vicinity of the LCST. We measured the temperature dependence of thermodiffusion and transformed these data sets into sigmoidal curves characterizing the phase transition of the polymer. Finite element modeling suggested a correction to the measured values that brought the transition temperatures measured by thermophoresis into accord with the cloud point curves. Our results demonstrate that observation of polymer thermodiffusion in a low volume dilute format is a facile method for determining polymer phase transition temperatures. [ABSTRACT FROM AUTHOR]

For the emergence of early life, the formation of biopolymers such as RNA is essential. However, the addition of nucleotide monomers to existing oligonucleotides requires millimolar concentrations. Even in such optimistic settings, no polymerization of RNA longer than about 20 bases could be demonstrated. How then could self-replicating ribozymes appear, for which recent experiments suggest a minimal length of 200 nt? Here, we demonstrate a mechanism to bridge this gap: the escalated polymerization of nucleotides by a spatially confined thermal gradient. The gradient accumulates monomers by thermophoresis and convection while retaining longer polymers exponentially better. Polymerization and accumulation become mutually self-enhancing and result in a hyperexponential escalation of polymer length. We describe this escalation theoretically under the conservative assumption of reversible polymerization. Taking into account the separately measured thermophoretic properties of RNA, we extrapolate the results for primordial RNA polymerization inside a temperature gradient in pores or fissures of rocks. With a dilute, nanomolar concentration of monomers the model predicts that a pore length of 5 cm and a temperature difference of 10 K suffice to polymerize 200-mers of RNA in micromolar concentrations. The probability to generate these long RNAs is raised by a factor of >10600 compared with polymerization in a physical equilibrium. We experimentally validate the theory with the reversible polymerization of DNA blocks in a laser-driven thermal trap. The results confirm that a thermal gradient can significantly enlarge the available sequence space for the emergence of catalytically active polymers. [ABSTRACT FROM AUTHOR]

The key requirement to solve the origin of life puzzle are disequilibrium conditions. Early molecular evolution cannot be explained by initial high concentrations of energetic chemicals since they would just react towards their chemical equilibrium allowing no further development. We argue here that persistent disequilibria are needed to increase complexity during molecular evolution. We propose thermal gradients as the disequilibrium setting which drove Darwinian molecular evolution. On the one hand the thermal gradient gives rise to laminar thermal convection flow with highly regular temperature oscillations that allow melting and replication of DNA. On the other hand molecules move along the thermal gradient, a mechanism termed Soret effect or thermophoresis. Inside a long chamber a combination of the convection flow and thermophoresis leads to a very efficient accumulation of molecules. Short DNA is concentrated thousand-fold, whereas longer DNA is exponentially better accumulated. We demonstrated both scenarios in the same micrometer-sized setting. Forthcoming experiments will reveal how replication and accumulation of DNA in a system, driven only by a thermal gradient, could create a Darwinian process of replication and selection. [ABSTRACT FROM AUTHOR]

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins. [ABSTRACT FROM AUTHOR]

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins. [ABSTRACT FROM AUTHOR]

The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method. [ABSTRACT FROM AUTHOR]

Thermophoresis is the movement of molecules in a temperature gradient. For aqueous solutions its microscopic basis is debated. Understanding thermophoresis for this case is, however, important since it proved very useful to detect the binding affinity of biomolecules and since thermophoresis could have played an important role in early molecular evolution. Here we discuss why the thermophoresis of single- and double-stranded oligonucleotides - DNA and RNA - is surprisingly similar. This finding is understood by comparing the spherical capacitor model for single-stranded species with the case of a rod-shaped model for double-stranded oligonucleotides. The approach describes thermophoresis of DNA and RNA with fitted effective charges consistent with electrophoresis measurements and explains the similarity between single- and double-stranded species. We could not confirm the sign change for the thermophoresis of single- versus double-stranded DNA in crowded solutions containing polyethylene glycol [Y. T. Maeda, T. Tlusty, and A. Libchaber, Proc. Natl. Acad. Sci. USA 109, 17972 (2012)], but find a salt-independent offset while the Debye length dependence still satisfies the capacitor model. Overall, the analysis documents the continuous progress in the microscopic understanding of thermophoresis. [ABSTRACT FROM AUTHOR]

Methods to move solvated molecules are rare. Apart from electric fields, only thermal gradients are effective enough to move molecules inside a fluid. This effect is termed thermophoresis, and the underlying mechanisms are still poorly understood. Nevertheless, it is successfully used to quantify biomolecule binding in complex liquids. Here we show experiments that reveal that thermophoresis in water is dominated by two electric fields, both established by the salt ions of the solution. A local field around the molecule drives molecules along an energy gradient, whereas a global field moves the molecules by a combined thermoelectrophoresis mechanism known as the Seebeck effect. Both mechanisms combined predict the thermophoresis of DNA and RNA polymers for a wide range of experimental parameters. For example, we correctly predict a complex, nonlinear size transition, a salt-species-dependent offset, a maximum of thermophoresis over temperature, and the dependence of thermophoresis on the molecule concentration. [ABSTRACT FROM AUTHOR]

Abstract: Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science. [Copyright &y& Elsevier]

The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac β1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine. [ABSTRACT FROM AUTHOR]