Handling of long DNA - applications and polymer physics

This thesis provides a proof of principle that deterministic lateral displacement (DLD) can be used to spatially separate DNA molecules by size. In order to achieve high separation quality, the pressure range, i.e. flow velocity has to be chosen carefully. The experiments were conducted in DLD devices fabricated in PDMS using soft lithography methods. Fluorescently labeled DNA molecules were transported through the device by pressure driven flow. The separation results were determined from and the separation result was recorded optically at the outlet. Both tested devices, with a critical diameter Dc of 0.64 μm and 0.75 μm respectively, showed capable of separating <10 kbp from 48.5 kbp DNA molecules with a separation quality of ~94%.DNA is the blueprint for life and contains all necessary information to ’build’ an organism. Therefore, DNA might be considered as the most important biologically relevant molecule. However, even though DNA is such a substantial molecule, the research methods and techniques regarding DNA are often surprisingly limited which is due to the complicated handling of the long molecule. This is also the case for DNA sorting methods, i.e the process of separating different DNA molecules from each other dependent on characteristic factors like weight or length. Sorting is especially important as pre-processing method for other established analysis techniques. Currently, the length-based sorting of DNA molecules is predominantly done using electrophoresis, for which a huge DNA sample volume with roughly a million molecules is required. Microfluidics is a relatively new, expanding field in biophysics which deals with fluids on the microliter and micrometer scale. Microfluidics are contrasted by macrofluidics which is our ’normal’ perception of fluids. An easy example which points out the difference is the surface tension of fluids. In every day life we experience it only as a side effect, but when dealing with cellular volumes, typically 1 μl which is one millionth of a liter, the surface tension wins over gravity. One application of microfluidics is the deterministic lateral displacement method, also called DLD, which was introduced by Richard Huang et al. in 2004. It is based on a micrometer-sized channel in which small obstacles are placed. If a fluid which also contains small particles flows through this obstacle-larded channel, the track of the particles vary dependent on their size: while very small particles follow the fluid flow and pass straight through the device, slightly larger particles get deflected to one of the channel walls. Thus, differently sized particles can be distinguished at the end of the channel as they get spatially separated. During the following master thesis, the sorting of DNA molecules dependent on their length was investigated. For this, the described DLD method was applied to DNA molecules in solution. It was found that DLD - if used under correct conditions - is an appropriate method to sort DNA molecules dependent on their length. This finding can be used in sample preparation and processing steps for DNA related experiments. Furthermore, since this is a microfluidic approach it allows DNA length separation for extremely small sample sizes, with only a couple of hundreds of molecules. Especially considering advancing single molecule analysis techniques and personalized medicine approaches, a cut-down sample volume will be crucial in future developments

tRNA sequences can assemble into a replicator

Can replication and translation emerge in a single mechanism via self-assembly? The key molecule, transfer RNA (tRNA), is one of the most ancient molecules and contains the genetic code. Our experiments show how a pool of oligonucleotides, adapted with minor mutations from tRNA, spontaneously formed molecular assemblies. They replicated information autonomously using only reversible hybridization under thermal oscillations. A pool of cross-complementary hairpins self-selected by agglomeration and sedimented under gravity. The metastable DNA hairpins bound to a template, consisting of one half of the hairpin assembly, and then interconnected by hybridization. Thermal oscillations separated replicates from their templates and drove an exponential, cross-catalytic replication. The molecular assembly could encode and replicate binary sequence information and reach a fidelity of 90 % per nucleotide. This mechanism of a replicating self-assembly of tRNA-like sequences indicates that the translation to proteins could be linked closer to molecular replication than previously thought

Selection of prebiotic oligonucleotides by cyclic phase separation

The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic processes such as daily temperature oscillations were ubiquitous in this environment and could trigger oligonucleotide phase separation. Here, we propose sequence selection based on phase separation cycles realized through sedimentation in a system subjected to the feeding of oligonucleotides. Using theory and experiments with DNA, we show sequence-specific enrichment in the sedimented dense phase, in particular of short 22-mer DNA sequences. The underlying mechanism selects for complementarity, as it enriches sequences that tightly interact in the condensed phase through base-pairing. Our mechanism also enables initially weakly biased pools to enhance their sequence bias or to replace the most abundant sequences as the cycles progress. Our findings provide an example of a selection mechanism that may have eased screening for the first auto-catalytic self-replicating oligonucleotides

Dual Lipolytic Control of Body Fat Storage and Mobilization in Drosophila

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. We generated a Drosophila mutant lacking the receptor (AKHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ß-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. Our data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis

DNA sample cleanup using deterministic lateral displacement

Optical mapping relies on the preparation of fluorescent DNA. DNA must be imaged with good signal to noise and therefore the background of unwanted DNA fragments, fluorescent dyes and other reagents need to be removed. We use deterministic lateral displacement to separate 48.5 kbp DNA from 50 kbp molecules from a background of shorter digested fragments. In both cases improving signal to noise during imaging

Heated gas bubbles enrich, crystallize, dry, phosphorylate and encapsulate prebiotic molecules

Non-equilibrium conditions must have been crucial for the assembly of the first informational polymers of early life, by supporting their formation and continuous enrichment in a long-lasting environment. Here, we explore how gas bubbles in water subjected to a thermal gradient, a likely scenario within crustal mafic rocks on the early Earth, drive a complex, continuous enrichment of prebiotic molecules. RNA precursors, monomers, active ribozymes, oligonucleotides and lipids are shown to (1) cycle between dry and wet states, enabling the central step of RNA phosphorylation, (2) accumulate at the gas-water interface to drastically increase ribozymatic activity, (3) condense into hydrogels, (4) form pure crystals and (5) encapsulate into protecting vesicle aggregates that subsequently undergo fission. These effects occur within less than 30 min. The findings unite, in one location, the physical conditions that were crucial for the chemical emergence of biopolymers. They suggest that heated microbubbles could have hosted the first cycles of molecular evolution