Structured sequences emerge from random pool when replicated by templated ligation.

The central question in the origin of life is to understand how structure can emerge from randomness. The Eigen theory of replication states, for sequences that are copied one base at a time, that the replication fidelity has to surpass an error threshold to avoid that replicated specific sequences become random because of the incorporated replication errors [M. Eigen, Naturwissenschaften 58 (10), 465-523 (1971)]. Here, we showed that linking short oligomers from a random sequence pool in a templated ligation reaction reduced the sequence space of product strands. We started from 12-mer oligonucleotides with two bases in all possible combinations and triggered enzymatic ligation under temperature cycles. Surprisingly, we found the robust creation of long, highly structured sequences with low entropy. At the ligation site, complementary and alternating sequence patterns developed. However, between the ligation sites, we found either an A-rich or a T-rich sequence within a single oligonucleotide. Our modeling suggests that avoidance of hairpins was the likely cause for these two complementary sequence pools. What emerged was a network of complementary sequences that acted both as templates and substrates of the reaction. This self-selecting ligation reaction could be restarted by only a few majority sequences. The findings showed that replication by random templated ligation from a random sequence input will lead to a highly structured, long, and nonrandom sequence pool. This is a favorable starting point for a subsequent Darwinian evolution searching for higher catalytic functions in an RNA world scenario.

Fission of Lipid-Vesicles by Membrane Phase Transitions in Thermal Convection.

Unilamellar lipid vesicles can serve as model for protocells. We present a vesicle fission mechanism in a thermal gradient under flow in a convection chamber, where vesicles cycle cold and hot regions periodically. Crucial to obtain fission of the vesicles in this scenario is a temperature-induced membrane phase transition that vesicles experience multiple times. We model the temperature gradient of the chamber with a capillary to study single vesicles on their way through the temperature gradient in an external field of shear forces. Starting in the gel-like phase the spherical vesicles are heated above their main melting temperature resulting in a dumbbell-deformation. Further downstream a temperature drop below the transition temperature induces splitting of the vesicles without further physical or chemical intervention. This mechanism also holds for less cooperative systems, as shown here for a lipid alloy with a broad transition temperature width of 8 K. We find a critical tether length that can be understood from the transition width and the locally applied temperature gradient. This combination of a temperature-induced membrane phase transition and realistic flow scenarios as given e.g. in a white smoker enable a fission mechanism that can contribute to the understanding of more advanced protocell cycles.

Proton gradients and pH oscillations emerge from heat flow at the microscale.

Proton gradients are essential for biological systems. They not only drive the synthesis of ATP, but initiate molecule degradation and recycling inside lysosomes. However, the high mobility and permeability of protons through membranes make pH gradients very hard to sustain in vitro. Here we report that heat flow across a water-filled chamber forms and sustains stable pH gradients. Charged molecules accumulate by convection and thermophoresis better than uncharged species. In a dissociation reaction, this imbalances the reaction equilibrium and creates a difference in pH. In solutions of amino acids, phosphate, or nucleotides, we achieve pH differences of up to 2 pH units. The same mechanism cycles biomolecules by convection in the created proton gradient. This implements a feedback between biomolecules and a cyclic variation of the pH. The finding provides a mechanism to create a self-sustained proton gradient to drive biochemical reactions.

Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip.

Cell adhesion processes are of ubiquitous importance for biomedical applications such as optimization of implant materials. Here, not only physiological conditions such as temperature or pH, but also topographical structures play crucial roles, as inflammatory reactions after surgery can diminish osseointegration. In this study, we systematically investigate cell adhesion under static, dynamic and physiologically relevant conditions employing a lab-on-a-chip system. We screen adhesion of the bone osteosarcoma cell line SaOs-2 on a titanium implant material for pH and temperature values in the physiological range and beyond, to explore the limits of cell adhesion, e.g., for feverish and acidic conditions. A detailed study of different surface roughness Rq gives insight into the correlation between the cells' abilities to adhere and withstand shear flow and the topography of the substrates, finding a local optimum at Rq = 22 nm. We use shear stress induced by acoustic streaming to determine a measure for the ability of cell adhesion under an external force for various conditions. We find an optimum of cell adhesion for T = 37 °C and pH = 7.4 with decreasing cell adhesion outside the physiological range, especially for high T and low pH. We find constant detachment rates in the physiological regime, but this behavior tends to collapse at the limits of 41 °C and pH 4.