DNA-Mediated Self-Assembly of Artificial Vesicles

Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time.In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts.This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and targeting of long-lived, pharmacologically inert prodrugs and their conversion to short-lived, active drug molecules directly at the site of action may be accomplished if multi-vesicle assemblies of predefined architecture are used

Formalizing modularization and data hiding in synthetic biology

Biological systems employ compartmentalization and other co-localization strategies in order to orchestrate a multitude of biochemical processes by simultaneously enabling “data hiding” and modularization. This article presents recent research that embraces compartmentalization and co-location as an organizational programmatic principle in synthetic biological and biomimetic systems. In these systems, artificial vesicles and synthetic minimal cells are envisioned as nanoscale reactors for programmable biochemical synthesis and as chassis for molecular information processing. We present P systems, brane calculi, and the recently developed chemtainer calculus as formal frameworks providing data hiding and modularization and thus enabling the representation of highly complicated hierarchically organized compartmentalized reaction systems. We demonstrate how compartmentalization can greatly reduce the complexity required to implement computational functionality, and how addressable compartments permit the scaling-up of programmable chemical synthesis

The MATCHIT automaton : exploiting compartmentalization for the synthesis of branched polymers

We propose an automaton, a theoretical framework that demonstrates how to improve the yield of the synthesis of branched chemical polymer reactions. This is achieved by separating substeps of the path of synthesis into compartments. We use chemical containers (chemtainers) to carry the substances through a sequence of fixed successive compartments. We describe the automaton in mathematical terms and show how it can be configured automatically in order to synthesize a given branched polymer target. The algorithm we present finds an optimal path of synthesis in linear time. We discuss how the automaton models compartmentalized structures found in cells, such as the endoplasmic reticulum and the Golgi apparatus, and we show how this compartmentalization can be exploited for the synthesis of branched polymers such as oligosaccharides. Lastly, we show examples of artificial branched polymers and discuss how the automaton can be configured to synthesize them with maximal yield

The Lagging Legs Exploiting Body Dynamics to Steer a Quadrupedal Agent

Abstract ⎯ The goal of this work was to steer a quadrupedal agent simply by changing the phase delay between its legs. Thus, we were able to show that a quadrupedal agent could possibly reach every point on a plane simply by exploiting its body dynamics. By exploiting body dynamics the controller has to fulfill only the function of a disturbance variable rather than exactly controlling the parameters of an agent’s movement. I

Experimental setup C: Implementation of a DNA-mediated vesicle-vesicle self-assembly.

<p>(A) Schematic representation of the result of experimental setup C (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009886#pone-0009886-g001" target="_blank">Figure 1</a>, setup C, panels e.i and e.ii). (B–E) Confocal laser scanning fluorescence (left) and differential interference contrast (right) micrographs in two columns of merged vesicle populations (VPs) C1 to C4 (VP C1 & VP C2, VP C3 & VP C4). For a detailed description of the experimental procedure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009886#pone-0009886-g001" target="_blank">Figure 1</a> (setup C). The biotinylated membranes (receptor surface density: 1.0 mol % biotin labeled PEG phospholipids) of all VPs were loaded with biotinylated single stranded DNA (biotin-ssDNA) using streptavidin as a cross-linking agent. VPs differed in streptavidin labeling (VPs C1/3: Alexa Fluor® 488, pseudocolored green in fluorescence micrographs, VPs C2/4: unlabeled) and biotin-ssDNA sequence (VP C1/3: α, VP C2: α', VP C4: β') – only sequences α and α' were complementary. Row headings indicate sodium iodide concentrations in the vesicle lumen and the surrounding medium. Fluorescence labeling of the membranes, silhouette blurring indicating vesicle lysis (cp. B.1, B.2), and accumulation of fluorescence signal positively correlate with sodium iodide concentration (microscope settings were identical for all pictures) causing a tradeoff between membrane loading, DNA hybridization, and vesicle stability. Adhesion plaques indicate stable vesicle-vesicle-linkage (visible adhesion plaques are highlighted in the differential interference contrast micrograph of D.1 by an image overlay with the confocal laser scanning fluorescence (processed) micrograph). The adhesion plaques of one vesicle (D.1) and DNA-independent vesicle-vesicle linkages (C.2) are highlighted by arrows. See text for a discussion of the loss of specificity of the DNA-mediated adhesion process observed in panels C.1 and C.2. Panel D.1 is reproduced with kind permission of Springer Science+Business Media (for original publication see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009886#pone.0009886-Hadorn2" target="_blank">[21]</a>). Scale bars represent 10 µm.</p

Programmability of the DNA-mediated self-assembly process: Multi-vesicle assemblies of predefined architecture.

<p>(A) Image overlays of confocal laser scanning fluorescence and differential interference contrast micrographs of merged vesicle populations (VPs). Biotinylated DNA single strands (biotin-ssDNA) that differ in sequence (α, α', β, β', γ, γ') and streptavidin populations that differ in fluorescence labeling (unlabeled (st.), Alexa Fluor® 488 labeled (st.-AF488, pseudocolored green in fluorescence micrographs), Alexa Fluor® 532 labeled (st.-AF532, pseudocolored red in fluorescence micrographs)) were incubated pairwise prior to vesicle decoration resulting in the following streptavidin/biotin-ssDNA combinations decorating the VPs: (A.1) α-st.-AF488: VP1, α'-st.:VP2; (A.2) α-st.-AF532 & β-st.-AF532: VP1, α'-st.-AF488 & γ-st.-AF488: VP2, β'-st. & γ'-st.: VP3. The receptor surface density was reduced to 0.25 mol % biotin labeled PEG phospholipids (cp. 1.0 mol % in experimental setup C). The fluorescence signal accumulates in the contact areas of adjacent and complementary vesicles forming adhesion plaques that indicate stable vesicle-vesicle linkage. (B) Schematic representation of the programmability of the DNA-mediated self-assembly process. The formation of adhesion plaques depends on the complementarity of ssDNA (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009886#pone-0009886-g004" target="_blank">Figure 4</a>.C.1) resulting in a sequence depend accumulation of linkers in the contact areas. The depletion of linkers in between the adhesion plaques terminates the self-assembly process. In combination with the ssDNA decoration of the vesicle surface, the self-termination defines the spatial arrangement of multi-vesicle aggregates. Thus, control of the assembly process is inherent to the system resulting either in duplets (A.1) or triplets (A.2) as minimal self-containing structural units. For a discussion of factors causing the low number of such units see text. Scale bars represent 10 µm.</p

Schematic representation of experimental setups A, B, and C.

<p>Numbers (0–3) indicate processes and small letters (a–e) indicate states. (Setup A) The incorporation of phospholipid-grafted PEG tethers into the vesicle membrane is analyzed. Vesicle populations (VPs) differ in the presence (VP A1) and absence (VP A2) of phospholipid-grafted fluorescently labeled PEG tethers (cfPEG2000-DSPE) during vesicle formation. (Setup B) To settle the specificity of membrane loading with streptavidin depending on the presence of anchoring sites, phospholipid-grafted biotinylated PEG tethers (bPEG2000-DSPE) are either present (VP B1) or absent (VP B2) during vesicle formation. Both VPs are subsequently incubated with fluorescently labeled streptavidin. Excess streptavidin is removed after incubation. (Setup C) To designate both the sequence-dependence and the dependence on the monovalent salt concentration of the vesicle self-assembly process two VPs either loaded with complementary (VP C1, VP C2) or noncomplementary (VP C3, VP C4) DNA single strands (ssDNA) are unified in solutions distinct in sodium iodide concentration. The streptavidin solutions were individually preincubated with biotin-ssDNA solutions prior to vesicle decoration (see microtubes holding the streptavidin/biotin-ssDNA solutions). After incubation of vesicles excess streptavidin/biotin-ssDNA is removed. DNA hybridization of complementary ssDNA causes accumulation of linkers and of the fluorescence signal (e.i) in the contact area over time (d to e) that is absent for noncomplementary ssDNAs (e.ii). (e.i) DNA-independent crystallization of streptavidin molecules on the surface of vesicles (*) that distributes stresses arising during/after DNA-mediated self-assembly may stabilize the linking system by compensating streptavidin molecules either incompletely equipped with biotin-ssDNA (**) or anchored only partially (***).</p

Experimental setup B: Streptavidin loading of the vesicle membrane in dependence of anchor sites concentration.

<p>(A) Schematic representation of the result of experimental setup B (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009886#pone-0009886-g001" target="_blank">Figure 1</a>, setup B, panels c.i and c.ii). (B) Confocal laser scanning fluorescence and (C) differential interference contrast micrographs of vesicle population (VP) B1 (left) and B2 (right). For a detailed description of the experimental procedure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009886#pone-0009886-g001" target="_blank">Figure 1</a> (setup B). VPs differed in the presence and absence of phospholipid-grafted biotinylated PEG tethers during vesicle formation. Both VPs were subsequently incubated with an excess of fluorescently labeled streptavidin (Alexa Fluor® 488, pseudocolored green in fluorescence micrographs). Scale bars represent 10 µm.</p