A genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice.

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

Cell-Free Gene Expression from DNA Brushes.

Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102-103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.

From deterministic to fuzzy decision-making in artificial cells.

Building autonomous artificial cells capable of homeostasis requires regulatory networks to gather information and make decisions that take time and cost energy. Decisions based on few molecules may be inaccurate but are cheap and fast. Realizing decision-making with a few molecules in artificial cells has remained a challenge. Here, we show decision-making by a bistable gene network in artificial cells with constant protein turnover. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow decision-making to a fuzzy and rapid regime dominated by small-number fluctuations. Gene regulation was observed at lower DNA and protein concentrations than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. The high-copy regime was characterized by a sharp transition and hysteresis, whereas the low-copy limit showed strong fluctuations, state switching, and cellular individuality across the decision-making point. Our results demonstrate information processing with low-power consumption inside artificial cells.

Control of lipid domain organization by a biomimetic contractile actomyosin cortex.

The cell membrane is a heterogeneously organized composite with lipid-protein micro-domains. The contractile actin cortex may govern the lateral organization of these domains in the cell membrane, yet the underlying mechanisms are not known. We recently reconstituted minimal actin cortices (MACs) (Vogel et al., 2013b) and here advanced our assay to investigate effects of rearranging actin filaments on the lateral membrane organization by introducing various phase-separated lipid mono- and bilayers to the MACs. The addition of actin filaments reorganized membrane domains. We found that the process reached a steady state where line tension and lateral crowding balanced. Moreover, the phase boundary allowed myosin driven actin filament rearrangements to actively move individual lipid domains, often accompanied by their shape change, fusion or splitting. Our findings illustrate how actin cortex remodeling in cells may control dynamic rearrangements of lipids and other molecules inside domains without directly binding to actin filaments.

Probing the Cooperativity of Binding Networks with High-Throughput Thermophoresis.

The formation of supramolecular complexes is found in many natural systems and is the basis for cooperative behavior. Here, we report on the development of a high-throughput platform to measure the complex binding behavior in 500 nL volumes and 1 536-well plates. The platform enabled us to elucidate the thermodynamic properties of a heterotrimeric DNA complex that portrays the structure of a biological relevant three-way junction. In a complementing set of cooperative networks, binding constants from ∼0.1 nM to ∼10 µM were measured by sampling a high-dimensional concentration space. Each intermediate binding state was probed simultaneously with only a single fluorescent label. Through systematic base pair variations, we observed the influence of the cooperative effect on single base pair mismatches. We further found coupled binding between seemingly independent binding sites through the complex structure of the three-way junction that could not have been observed without the measurement of the entire network. These results promote automated high-throughput thermophoresis to characterize arbitrary binding networks.

Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy.

In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 µm(2)/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 µm(2)/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms.

Understanding the similarity in thermophoresis between single- and double-stranded DNA or RNA.

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.