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Reaction–Diffusion Patterning of DNA-Based Artificial Cells

  • Adrian Leathers
    Adrian Leathers
    Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
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  • , 
  • Michal Walczak
    Michal Walczak
    Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
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    Orcidhttps://orcid.org/0000-0002-4701-9476
  • , 
  • Ryan A. Brady
    Ryan A. Brady
    Department of Chemistry, Faculty of Natural and Mathematical Sciences, King’s College London, London SE1 1DB, U.K.
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    Orcidhttps://orcid.org/0000-0002-0408-3224
  • , 
  • Assala Al Samad
    Assala Al Samad
    Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, U.K.
    Department of Chemistry, University College London, London WC1H 0AJ, U.K.
    More by Assala Al Samad
  • , 
  • Jurij Kotar
    Jurij Kotar
    Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
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  • , 
  • Michael J. Booth
    Michael J. Booth
    Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, U.K.
    Department of Chemistry, University College London, London WC1H 0AJ, U.K.
    More by Michael J. Booth
    Orcidhttps://orcid.org/0000-0002-4224-798X
  • , 
  • Pietro Cicuta
    Pietro Cicuta
    Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
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    Orcidhttps://orcid.org/0000-0002-9193-8496
  • , and 
  • Lorenzo Di Michele*
    Lorenzo Di Michele
    Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, London W12 0BZ, U.K.
    fabriCELL, Imperial College London, Molecular Sciences Research Hub, London W12 0BZ, U.K.
    Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
    *Email: [email protected]
    More by Lorenzo Di Michele
    Orcidhttps://orcid.org/0000-0002-1458-9747
Cite this: J. Am. Chem. Soc. 2022, 144, 38, 17468–17476
Publication Date (Web):September 14, 2022
https://doi.org/10.1021/jacs.2c06140

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Abstract

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Biological cells display complex internal architectures with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts to create advanced cell mimics, namely, artificial cells, demands strategies for constructing similarly heterogeneous structures with localized functionalities. Here, we introduce a platform for constructing membraneless artificial cells from the self-assembly of synthetic DNA nanostructures in which internal domains can be established thanks to prescribed reaction–diffusion waves. The method, rationalized through numerical modeling, enables the formation of up to five distinct concentric environments in which functional moieties can be localized. As a proof-of-concept, we apply this platform to build DNA-based artificial cells in which a prototypical nucleus synthesizes fluorescent RNA aptamers that then accumulate in a surrounding storage shell, thus demonstrating the spatial segregation of functionalities reminiscent of that observed in biological cells.

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Introduction

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Bottom-up synthetic biology aims to engineer artificial systems that exhibit biomimetic structures and functionalities via the rational combination of molecular and nanoscale elements. These systems often take the form of artificial cells (ACs), microrobots constructed de novo to replicate a subset of the behaviors typically associated with biological cellular life, including communication, adaptation, energy conversion, and motility. (1−3) Despite still being far from the complexity of live cells, ACs are regarded as promising technological platforms for personalized healthcare, where cell-like microdevices could operate in vivo, detect disease-related biomarkers, and respond by synthesizing and releasing therapeutic agents, potentially resulting in minimally toxic and efficient treatments. (1,4−7) Similarly, ACs could underpin innovations in synthesis, through the optimized production of materials and pharmaceuticals and in environmental remediation by selectively capturing and storing pollutants. (1,4,5,8,9)
ACs often rely on microcompartments constructed from lipid, (10,11) polymer, (11,12) or protein membranes, (13) but membraneless implementations based on coacervates (14−19) or hydrogels (18−21) are gaining traction, which is driven by their enhanced robustness and easy manufacture as well as the renewed interest in biomolecular condensates and membraneless compartments in cell biology. (22,23) Similar to the case of biological condensates, the accumulation of target molecules within membraneless ACs can be induced through selective affinity for the scaffold phase without relying on a membrane.
Like (eukaryotic) cells, AC implementations can benefit from a heterogeneous internal architecture that regulates the transport and spatial distribution of (bio)molecules, which can facilitate the design of biomimetic pathways that require the colocalization or separation of specific compounds. (24) However, while with membrane-based platforms it is relatively easy to establish internal heterogeneity, for instance, through nesting or sequential assembly, (24) no general platform has been proposed to program the local composition in membraneless scaffolds. Here we leverage the structural and dynamic programmability afforded by DNA nanotechnology (25,26) to construct membraneless condensates of DNA nanostructures, which can be “patterned” thanks to a reaction–diffusion scheme (27−30) (Figure 1a). This strategy can generate up to five chemically addressable, distinct microenvironments in a concentric geometry, whose features can be rationalized through numerical modeling. As a proof-of-concept, we use the platform to create model ACs with a spatially resolved functionality, namely, where a fluorescent RNA aptamer is synthesized in a prototypical “nucleus” and accumulates in an outer shell (Figure 1a, right).

Figure 1

Figure 1. Reaction–diffusion patterning of amphiphilic DNA condensates. (a) Reaction–diffusion processes are used to pattern initially uniform DNA condensates and construct DNA-based artificial cells featuring distinct internal environments of controllable number and molecular makeup, unlocking the spatial engineering of functionality. (b) Foundational building block of the condensates, consisting of a locked four-way DNA junction with cholesterol moieties at the end of each arm. The constructs are composed of four distinct strands that form the junction (blue) and four identical cholesterolized oligonucleotides (orange). (31−33) One arm features an additional overhang connected to a base strand (b), which serves as a binding site for complementary freely diffusing patterning strands. The latter range between 16  (p1) and 40 nt (p8) in length and compete over (color-coded) overlapping binding domains on the base strand. p1 and p8 shown here are functionalized with Alexa 594 (red) and Alexa 488 (green) fluorophores, respectively. The stop strand (s) has the same sequence as the base strand and can be added in solution to sequester the excess patterning strands. Sequences of all the DNA oligonucleotides are provided in Table S1. (c) Assembly process for amphiphilic DNA condensates. Samples containing all single-stranded DNA components are slowly annealed from 90 to 20 °C, leading to the formation of a nanoporous framework in which the branched DNA motifs connect micelle-like hydrophobic cores where the cholesterol modifications localize, as previously reported. (31−33) Sample preparation details are provided in the Experimental Methods (SI). (d) Schematic depiction of the designed reaction–diffusion pathway. At time t = 0, condensates are exposed to a solution of p1 (short, red) and p8 (long, green) patterning strands in excess concentrations compared to the base strands. Short p1 DNA strands are able to diffuse inside the condensates faster than long p8 strands, allowing for prior binding to the base strand (red box). At later times, p8 strands then diffuse within the condensates and, due to the sequence design, are able to displace p1 strands via toeholding (36,37) (green box). The result is a sequence of two fronts that propagate inward through the condensate. (e) Series of confocal micrographs of the process discussed in panel c, where propagating fronts are visualized thanks to fluorescent modifications of p1 and p8. the scale bar represents 15 μm.

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Results and Discussion

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Our condensates self-assemble from branched amphiphilic DNA nanostructures, as shown in Figure 1b. Similar constructs were previously demonstrated to form nanoporus phases with programmable structures, molecular-sieving properties, stimuli responsiveness, and the ability to host dynamic DNA circuitry. (31−35) As depicted in Figures 1c and S1 and detailed in the Experimental Methods (SI), spherical aggregates with cell-like dimensions (10–40 μm in diameter) readily emerge from a one-pot annealing reaction. Small-angle X-ray scattering measurements demonstrate that the condensates display internal crystalline order with a BCC unit cell and lattice parameter of 26.8 nm, which is consistent with previous observations on similar systems (Figure S2). (32)
The foundational building block used here consists of a DNA four-way junction, where the end of each 35 base-pair (bp) duplex arm is label by cholesterol moieties (Figure 1b). One of the arms was modified with an additional single-stranded overhang to which a base (b) strand was connected. The base strand serves as a competitive binding site for freely diffusing patterning strands (p) of different lengths, as depicted in Figure 1b, where the complementarity of domains is shown by the same coloration and opposite directionality. All the patterning strands feature an identical (red) domain complementary to the base, but for longer strands the complementarity is extended to more adjacent domains. This feature allows any longer patterning strand to displace a shorter strand from the base via toehold-mediated strand displacement (toeholding) (36,37) but not vice versa, establishing a length-dependent binding hierarchy. The length of even the shortest binding domain (14 nucleotides (nt)) is such that the thermal detachment of the patterning strands does not occur within experimentally relevant time scales. Sequences of all oligonucleotides are reported in Table S1.
The principle for AC patterning is schematically depicted in Figure 1d. Condensates are prepared hosting uniformly distributed base strands but without any initially connected patterning strands. Multiple types of patterning strands are then introduced in solution in excess concentrations compared to the number of available binding sites in the condensates (see Experimental Methods (SI)). In this example, we introduce patterning strands p1 (16 nt) and p8 (40 nt) labeled with an Alexa 594 (red) and Alexa 488 (green), respectively. Note that we label patterning strands with numbers that increase with the strand length such that p(n + 1) is longer than pn. The diffusion coefficient of DNA decreases with the contour length. (38) This dependency is enhanced in porous environments like our condensates, (39) allowing shorter DNA strands to diffuse significantly faster than longer ones. The shorter patterning strands, p1 in the example, will thus rapidly access the condensate, occupying base strands progressively from the outside of the condensate inward. At later times, the longer p8 strands also diffuse through the condensate and, as they do so, displace p1 strands from the base strands via toeholding and release them back into solution. The result, experimentally demonstrated through confocal micrographs in Figure 1e, is a sequence of inward-propagating fluorescent fronts: a red wave corresponding to the rapidly diffusing p1 appears first, which is then replaced by a green front produced by the slowly diffusing but strongly binding p8 strands. Note that in confocal measurements the signal from excess patterning strands in solution is not visible due to the comparatively much higher concentration of binding sites within the condensates.
This scheme thus allows us to localize different oligonucleotides in distinct and individually addressable concentric shells within the condensates. While in this example the patterning strands bear a simple fluorescent modification, one could easily envisage the inclusion of functional elements, thus paving the way to establishing a spatially resolved functionality in membraneless ACs.
Domain structure and evolution can be programmed via the number and length of patterning strands, as summarized in Figure 2. Confocal micrographs for representative condensates exposed to one, two, three, and five patterning strands are shown in Figure 2a–d-ii, while Videos S1–S12 exemplify pattern evolution in individual condensates and larger sample areas (see the supplementary videos key in the SI). In Figure 2a–d-iii, the spatiotemporal evolution of the patterns is captured by 2D color maps showing the (azimuthally averaged and normalized) radial profile of the fluorescence intensity, I(r, t), where r is the distance from the centroid of the condensate and t is the time elapsed from exposure to the patterning strands (see Experimental Methods (SI)). Examples of analogous color maps for multiple condensates and different numbers of patterning strands are shown in Figures S3–6. For tests with more than two domains, nonfluorescent (dark) patterning strands of lengths intermediate to the two fluorescent ones were used. For instance, as shown in Figure 2c, a dark 30 nt strand (p6) is used in combination with 16 nt (red) p1 and 40 nt (green) p8, generating a dark shell that separated the fast-propagating red wave and the slow-propagating green wave. Two dark (p3 and p7) and three fluorescent strands (p1, p5, and p8) are used in Figure 2d, generating five distinct microenvironments at ∼7 min from exposure to the patterning strands, as highlighted by the azimuthally averaged fluorescent intensity profiles (Figure 2d-iv). Note that in this case the difference in length between adjacent species varies between 5 and 7 nt, demonstrating a separation ability comparable to those of electrophoretic techniques and hinting at the possible applications of ACs in the detection and separation of nucleic acids. For a fixed number of patterning strands, the relative widths of the domains can be controlled by the design, as shown in Figure S7 for three-domain experiments in which dark strands of different lengths were used.

Figure 2

Figure 2. Condensate patterning is predictable and customizable. (a–d) Patterning-strand scheme (-i) and equatorial confocal microscopy sections (-ii) for condensates patterned to form an increasing number of concentric domains, from one in system a to five in system d. Some patterning strands are fluorescently labeled with Alexa 594 (p1) and Alexa 488 (p5 and p8) while others do not bear modifications, resulting in dark regions intermitting the fluorescent shells in the confocal data. See Table S1 for the DNA sequences. The spatiotemporal evolution of the domain structure is visualized as the azimuthally averaged, normalized radial intensity profile I(r, t), where r is the radial coordinate defined from the centroid of the condensate and t is the time elapsed from exposure of the condensates to the patterning strands (-iii). For systems a–c, I(r, t) is compared with the fitted outcome of a reaction–diffusion numerical model (-iv). Note that early times are not shown in experimental color maps (gray bands) due to a delay between the time at which condensates were exposed to the patterning strand (t = 0) and the start of the confocal recording. See the Experimental Methods (SI) for information on image analysis and numerical modeling. For system d, subpanel d-iv shows the radial intensity profiles extracted from confocal images at t = 7 min, highlighting the presence of five distinct domains. The green dotted and red dashed lines mark the signals from the Alexa 488 (p5 and p8) and Alexa 594 (p1) channels, respectively, while the black solid line represents the overall intensity. All profiles are normalized by their highest value. (e) Domain propagation can be arrested by adding an excess of the stop strand (s) in solution (e-i, see also Figure 1a), as demonstrated in e-ii with confocal data for a system with three patterning strands (p1, p6, and p8). The stop strand was added at t = 16 min, after which no further pattern evolution was observed (besides photobleaching). Videos S1–S8 show the pattern evolution in individual condensates (even numbered) and larger fields of view (odd numbered). See the supplementary videos key in the SI. Scale bars represent 15 μm.

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In all examples discussed, the reactions are designed to progress toward the equilibrium configuration in which the longest, most stable construct occupies all binding sites, defying the purpose of our strategy as a means of engineering an internal AC architecture. However, pattern evolution can be readily arrested using a stop strand (s, Figure 1a), with a sequence identical to that of the base. The stop strand is added in solution in an excess concentration compared to that of all patterning strands combined (see Experimental Methods (SI)), sequestering them and interrupting wavefront propagation. Figure 2e-ii shows that patterns arrested with this protocol remained stable for several hours, as required for the purpose of spatial engineering in ACs. The lack of any visible blurring of the patterns confirms the absence of internal diffusion of the amphiphilic DNA building blocks that make up the structure of the condensates.
The system’s evolution can be modeled through a set of coupled reaction–diffusion equations under the assumptions of a spherical condensate geometry and an excess of patterning strands in solution, as fully detailed in the Modeling Methods (SI). Alongside known or easily determined system parameters such as condensate size, the model requires as inpust the diffusion constants (D) of the patterning strands, the second-order rate constants through which the patterning strands bind the base or displace previously bound strands (kon), and the exchange rate of patterning strands between the bulk and the condensate (kin). (40,41) The latter three quantities (D, kon, and kin), which are graphically depicted in Figure 3a, are used as fitting parameters. The model also features a partition coefficient of the patterning strands within the condensates; (40) for realistic values, this coefficient was found to have no significant effect on the fitting outcomes and was thus set to 1 (Figure S8).

Figure 3

Figure 3. Model fitting enables the extraction of reaction–diffusion parameters. (a) Schematic representation of the color parameters, which consist of an entry rate kin, a diffusion constant D, and a binding or displacement rate kon (top). With kon, we indicate both the second-order binding rate of a patterning strand to a free binding site and that of the toehold-mediated strand displacement process through which a longer patterning strand replaces a shorter one that previously occupied a binding site (bottom, see the Modeling Methods (SI). (b and c) Diffusion coefficients for the 40 nt patterning strand p8 and the 16 nt patterning strand p1, respectively. (d and e) Binding rates for the 40 nt patterning strand p8 and the 16 nt patterning strand p1, respectively. Data are shown for samples with one patterning strand (p1 or p8; Figures 2a, S3, and S4; N = 33 condensates for p1 and N = 29 condensates for p8), two patterning strands (p1 and p8; Figures 2b and S5; N = 23 condensates) and three patterning strands (p1, p6, and p8; Figures 2c and S6; N = 43 condensates). The results are displayed as box plots with highlighted median, upper, and lower quartiles (box); 50th centile (whiskers) outliers are excluded. Overlaid on the box plots are the means (symbol) and standard deviations (error bar the same color as the symbol) of the distributions.

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The model outputs the spatiotemporal evolution of the concentration of bound patterning strands within the condensates, which, after accounting for diffraction-induced blurring and normalization, renders an estimate of the experimental fluorescent intensities. This model can thus be used to fit the experimental I(r, t) data for up to three competing patterning strands (see Modeling Methods (SI)). Qualitatively comparing the experimental and fitted I(r, t) maps demonstrates their good agreement, as can be seen in Figures 2a–-c-iv and S3–S6. A quantitative assessment of the fit residuals, shown in Figure S9, confirms the good match, with deviations typically within ±10–15% for data associated with long (p8) patterning strands and within ±20–25% for short (p1) strands. The larger deviation observed for the shorter patterning strands can be ascribed to the smaller data sets given that short strands experience a shorter reaction–diffusion transient. In Figure S10 we further show histograms of the residuals that combine values from all sampled condensates. The distributions appear quite symmetrical but deviate from a normal profile. A non-normal distribution may result from the small shape differences between the modeled and experimental reaction–diffusion profiles, which is also highlighted in the residual maps (Figure S9). These differences may emerge due to early time effects related to sample mixing, optical artifacts due to refractive index mismatches and absorption, nonspherical condensate geometry, or nonisotropic diffusion caused by contact with the bottom of the experimental chamber.
Panels b and c of Figure 3 show the distributions of fitted diffusion constants and binding coefficients, respectively, for our shortest (p1, 16 nt) and longest (p8, 40 nt) patterning strands, as determined for experiments with a single patterning strand (p1 or p8; Figures 2a, S3, and S4), two patterning strands (p1 and p8; Figures 2b and S5), and three patterning strands (p1, p6, and p8; Figures 2c and S6).
We observe an order-of-magnitude difference in the diffusion constant between p1 and p8, with Dp1 = 1–4 × 10–7  cm2 s–1 and Dp8 = 1–4 × 10–8  cm2 s–1. D is the primary parameter that determines the propagation speed of the reaction–diffusion fronts through the condensates; therefore, the difference found between Dp1 and Dp8 is consistent with expectations. We further note that both diffusion constants decrease in the presence of additional patterning strands, hinting at crowding effects.
Panels d and e in Figure 3 show the fitting outcomes for the rate constant kon. In all cases, given that p1 is unable to displace other incumbent strands, kon,p1 describes the binding of p1 to a free base strand. Similarly, kon,p8 describes hybridization to free base strands in experiments only featuring p8. When shorter patterning strands are present alongside p8, kon,p8 can be interpreted as the effective second-order rate constant of the toehold-mediated strand displacement reaction through which p8 displaces incumbent p1 (two-strand case) and p6 (three-strand case). (36) See Figure 3a (bottom) and Modeling Methods (SI) for a quantitative justification of this interpretation. Fits produce values of kon,p1 and kon,p8 within the same order of magnitude (104–105 M–1 s–1), with the former slightly larger than the latter. Values are consistent with previous observations for hybridization in hydrogels (28) and smaller than rates typically measured in freely diffusing constructs (∼106 M–1 s–1). (36) The similarity between the rates of hybridization (kon,p1) and toeholding (kon,p8) is expected given that the two are known to converge for toehold lengths in excess of 6 nt, a condition that is verified in our reactions. (36) A decreasing trend was found for an increasing number of patterning strands, which was more pronounced for kon,p8 compared to kon,p1 and may thus be due to crowding.
The material exchange rate kin, as summarized in Figure S11, is slightly larger for p1 compared with p8, which is consistent with the higher diffusion rate of the shorter patterning strand. Meanwhile, no significant trend was observed as a function of the number of patterning strands.
To explore possible correlations between the fitting parameters and to gauge the robustness of our fits, we computed maps of the sum of squared residuals, χ2, as a function of each pair of parameters (Figure S12). (42−44) The maps reveal a degree of correlation between D and kin, while kon is not correlated to either variable. All maps, however, show a clear minimum, hinting at the identifiability of all parameters, which was confirmed by the individual likelihood curves and associated identifiability analysis (Figure S13). We note that while kon is identifiable, the model displays a relatively weak sensitivity to this parameter for values in excess of ∼105 M–1 s–1 (Figures S12a and S13a). This weakened sensitivity can be rationalized as the result of the diffraction blurring applied in the model, which for sufficiently large values of kon dominates over the blurring of the propagating front induced by finite reaction rates (see Modeling Methods (SI) for details).
Having identified a route for establishing addressable domains in condensates, we can proceed with localizing functional elements within different regions to create a model AC that hosts a spatially organized pathway. As summarized in Figure 4a, the AC was patterned with a “nucleus” region hosting a template construct (t), which contains a T7 RNA polymerase promoter sequence and the DNA sequence complementary to an RNA aptamer, and the shell region containing binding sites for the RNA product. The template is anchored to the base strand through a bridging (r) strand, which ensures the formation of the double-stranded T7 promoter required for the T7 polymerase to begin transcription. (45) The RNA produced was a modified version of the DFHBI-binding fluorescent Broccoli aptamer. (46) Addition of an extra 8 bps to the stem region produced a significantly brighter aptamer, as discussed in the Experimental Methods (SI) and Figure S14. The aptamer also features a binding site complementary to the single-stranded overhang present in the capture (c) strands located in the shell region. Note that because the r–t complex located in the nucleus is longer than the capture strand present in the shell, the patterning of these devices needed to be conducted following a multistep protocol, as detailed in the Experimental Methods (SI) and Figure S15. The protocol also involves washing steps to remove any unbound r–t complexes that would result in RNA synthesis occurring in the bulk solution.

Figure 4

Figure 4. Spatially distributed functionality in a model artificial cell. (a) Schematics of the functional nucleic acid machinery in the nucleus (cyan) and shell regions (orange). In the nucleus, connected to the base strand are a bridge (r) strand and the template (t) strand. Together, these form a double-stranded T7 promoter (pink) and a single-stranded polymerase template (purple, red) from which a polymerase (black) is able to synthesize Broccoli RNA aptamers (folded purple and red). These aptamers then form a complex with DFHBI molecules to become fluorescent (orange). The base strands in the shell region are connected to capture strands (c) with single-stranded overhangs (red) complementary to a free domain on the broccoli aptamer. Complementary DNA and RNA domains are shown in the same color. Protocols for patterning the ACs are detailed in the Experimental Methods (SI). (b) Mode of operation of the AC. The polymerase is added in solution alongside NTPs, DFHBI, and other components required for Broccoli synthesis, which diffuse through the shell (1) to reach the nucleus, where the aptamers are produced (2). The aptamers then diffuse outward and bind to the dedicated sites in the shell (3). (c) A series of confocal images (top) of an AC progressively building the Broccoli aptamer in the shell (orange). Note how the signal accumulates from the nucleus–shell interface and propagates outward. The nucleus is shown in cyan and progressively photobleaches. The dashed lines mark the physical boundary of the AC and that between the nucleus and the shell. The bottom images of bright-field images of the same AC overlaid onto (faint) confocal data, demonstrating that no physical change to the AC occurs during Broccoli synthesis. The reaction is initiated at time t = 0, as discussed in the Experimental Methods (SI). (d) Color map showing the evolution of the radial fluorescent intensity of the aptamer. The slope of the fluorescent front signals accumulation from the inside out, as highlighted by the white arrow. Dashed lines mark the nucleus–shell and shell/–olution boundaries. Videos S13–S16 show the responses of multiple ACs with different shell–nucleus size ratios. The scale bar represents 15 μm.

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The sought response at the AC level is sketched in Figure 4b. Patterned ACs are exposed to a polymerase that, similar to other proteins of comparable size, (32) can diffuse through the condensates, reaching the template in the nucleus. Here, the Broccoli aptamer, which readily binds to DFHBI, is produced, diffuses out toward the shell, and binds the capture motifs. These constructs would thus display the envisaged separation of functionality in addition to a basic form of communication between two domains, one producing a signal in the form of RNA constructs and the other receiving it.
Figure 4c (top) shows a time-resolved sequence of confocal micrographs from an AC that produced the designed response. The nucleus (cyan), fluorescently stained thanks to a fluorophore on the bridging strand, remained the same size through the experiment and only underwent progressive bleaching. In turn, fluorescence from the Broccoli aptamer (orange) builds up in the shell region from the inside out, consistent with the RNA product being produced in the nucleus. Combined bright-field and confocal micrographs of the same objects confirm that the overall size and appearance of the condensate does not change during Broccoli accumulation (Figure 4c, bottom). A color map of the radial intensity of the Broccoli emission versus time is shown in Figure 4d, where the outward-propagating front, marked by an arrow, is clearly observable. Data from more ACs with different shell thicknesses relative to nucleus size are summarized in Figure S16, while time lapses of the process can be inspected in Videos S13–S16. As a control, in Figure S17 we show the results of experiments with template or promoter constructs free in solution, which as expected show outside-in accumulation. Finally, in Figures S18 and S19 we report the time-dependent Broccoli emission in bulk fluorimetry experiments, including samples with aptamer-expressing ACs and samples containing only the supernatant solution but no ACs. The lack of signal from the latter further confirms that aptamer production occurs exclusively within the AC nucleus.

Conclusion

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In summary, we have introduced a general process for the creation of stable and individually addressable domains in condensates self-assembled from DNA nanostructures. The process relies on a reaction–diffusion scheme, where patterning constructs with different diffusivities and binding affinities compete for binding sites within the condensates. The number and size of the domains can be tuned by design and predicted by numerical modeling. As a proof-of-concept, we adapted the patterning scheme to construct a model DNA-based artificial cell with an active nucleus that produced a fluorescent RNA aptamer and a storage shell where the product progressively accumulated. With this basic implementation as a starting point, one could envision future developments toward artificial cells capable of producing, storing, and later releasing therapeutic RNA elements, such as small-interfering RNAs. (47) These implementations, however, would need to also encapsulate the RNA polymerase and NTPs rather than relying on freely diffusing enzymes and building blocks, which could be achieved by surrounding the artificial cell with a less permeable barrier.
More generally, we argue that reaction–diffusion-patterned DNA condensates could constitute a versatile platform for engineering cell-like agents with spatially- and temporally resolved functionalities. Potential responses are not limited to the expression or capture of RNA products, as the domains can be enriched with virtually any functional molecule or nanoscale agent that can be linked to the patterning strands, including enzymes, nanoparticles, aptamers, and photoresponsive elements, thus unlocking the opportunity to engineer evermore complex reaction pathways within the ACs. Finally, while here DNA condensates are used as passive scaffolds, one can envisage design modifications where patterning alters the local physical structure. For instance, one could ensure that the localization of responsive moieties makes specific regions in the condensates sensitive to targeted degradation (photoinduced or enzymatic), enabling the creation of voids within the artificial cells that could be used to store bulky cargoes or simply regulate the connectivity of the remaining domains. In turn, dynamic changes in the local mesh size could enable the design of pathways that couple changes in local transport properties with biochemical activity, giving rise to more complex time-dependent responses. For example, one could envisage a negative feedback loop where the local accumulation of an RNA product represses transcription by (sterically) hindering polymerase diffusion and configurational freedom. If coupled with enzymatic RNA degradation, similar systems could sustain fluctuations in RNA expression periodic in time and space, reminiscent of the oscillating gene expression in biological cells. (48) Similarly, RNA expression could be coupled to changes in the artificial cell size, leading to mechanical actuation useful for engineering propulsion or shape changes in artificial tissues.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c06140.

  • All experimental, image analysis, numerical modeling and fitting methods; bright-field microscopy images; SAXS characterizations; I(r, t) patterning maps; data demonstrating control over domain thickness; analysis of the fitting model behavior; correlation between fitting parameters and their identifiability; performance of the extended Broccoli aptamer; patterning protocol for the RNA-synthesizing ACs; RNA synthesis in ACs; control experiments; nuclotide sequences; and supplementary video descriptions (PDF)

  • Large view of one patterning strand (p8) (AVI)

  • Zoomed-in view of one patterning strand (p8) (AVI)

  • Large view of two patterning strands (p1 and p8)(AVI)

  • Zoomed-in view of two patterning strands (p1 and p8)(AVI)

  • Large view of three patterning strands (p1, p6, and p8) (AVI)

  • Zoomed-in view of three patterning strands (p1, p6, and p8) (AVI)

  • Large view of five patterning strands (p1, p3, p5, p7 and p8) (AVI)

  • Zoomed-in view of five patterning strands (p1, p3, p5, p7 and p8)(AVI)

  • Large view of three patterning strands (p1, p6, and p8) and the stop strand (AVI)

  • Zoomed-in view of three patterning strands (p1, p6, and p8) and the stop strand (AVI)

  • Large view of one patterning strand (p1) (AVI)

  • Zoomed-in view of one patterning strand (p1) (AVI)

  • Broccoli RNA production and storage in artificial cells (example 1) (AVI)

  • Broccoli RNA production and storage in artificial cells (example 2) (AVI)

  • Zoomed-in view of Broccoli RNA production and storage in artificial cells (fluorescence) (AVI)

  • Zoomed-in view of Broccoli RNA production and storage in artificial cells (bright-field and light fluorescence) (AVI)

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  • Corresponding Author
    • Lorenzo Di Michele - Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, London W12 0BZ, U.K.;  fabriCELL, Imperial College London, Molecular Sciences Research Hub, London W12 0BZ, U.K.;  Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.;  Orcidhttps://orcid.org/0000-0002-1458-9747;  Email: [email protected]
  • Authors
    • Adrian Leathers - Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
    • Michal Walczak - Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.;  Orcidhttps://orcid.org/0000-0002-4701-9476
    • Ryan A. Brady - Department of Chemistry, Faculty of Natural and Mathematical Sciences, King’s College London, London SE1 1DB, U.K.;  Orcidhttps://orcid.org/0000-0002-0408-3224
    • Assala Al Samad - Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, U.K.;  Department of Chemistry, University College London, London WC1H 0AJ, U.K.
    • Jurij Kotar - Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
    • Michael J. Booth - Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, U.K.;  Department of Chemistry, University College London, London WC1H 0AJ, U.K.;  Orcidhttps://orcid.org/0000-0002-4224-798X
    • Pietro Cicuta - Biological and Soft Systems, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.;  Orcidhttps://orcid.org/0000-0002-9193-8496
  • Notes
    The authors declare no competing financial interest.

    A dataset associated to this publication is available free of charge at https://doi.org/10.17863/CAM.87545.

Acknowledgments

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L.D.M. acknowledges support from a Royal Society University Research Fellowship (UF160152) and from the European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (ERC-STG No 851667 – NANOCELL). A.L. and L.D.M. acknowledge support from a Royal Society Research Grant for Research Fellows (RGF/R1/180043). M.J.B. is supported by a Royal Society University Research Fellowship (URF/R1/180172). M.J.B. and A.A.S. acknowledge funding from a Royal Society Enhancement Award (RGF/EA/181009) and an EPSRC New Investigator Award (EP/V030434/1). M.W. acknowledges support from the Engineering and Physical Sciences Research Council (EPSRC) and the Department of Physics at the University of Cambridge (the McLatchie Trust fund). The authors acknowledge Diamond Light Source for providing synchrotron beamtime (SM28071) and thank A. Smith for assistance in operating beamline I22.

References

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    Qiao, Yan; Li, Mei; Booth, Richard; Mann, Stephen
    Nature Chemistry (2017), 9 (2), 110-119CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Recent progress in the chem. construction of colloidal objects comprising integrated biomimetic functions is paving the way towards rudimentary forms of artificial cell-like entities (protocells). Although several new types of protocells are currently available, the design of synthetic protocell communities and investigation of their collective behavior has received little attention. Here we demonstrate an artificial form of predatory behavior in a community of protease-contg. coacervate microdroplets and protein-polymer microcapsules (proteinosomes) that interact via electrostatic binding. The coacervate microdroplets act as killer protocells for the obliteration of the target proteinosome population by protease-induced lysis of the protein-polymer membrane. As a consequence, the proteinosome payload (dextran, single-stranded DNA, platinum nanoparticles) is trafficked into the attached coacervate microdroplets, which are then released as functionally modified killer protocells capable of rekilling. Our results highlight opportunities for the development of interacting artificial protocell communities, and provide a strategy for inducing collective behavior in soft matter microcompartmentalized systems and synthetic protocell consortia.
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  17. 17
    Samanta, A.; Sabatino, V.; Ward, T. R.; Walther, A. Functional and morphological adaptation in DNA protocells via signal processing prompted by artificial metalloenzymes. Nat. Nanotechnol. 2020, 15, 914– 921,  DOI: 10.1038/s41565-020-0761-y
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    17
    Functional and morphological adaptation in DNA protocells via signal processing prompted by artificial metalloenzymes
    Samanta, Avik; Sabatino, Valerio; Ward, Thomas R.; Walther, Andreas
    Nature Nanotechnology (2020), 15 (11), 914-921CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
    Abstr.: For life to emerge, the confinement of catalytic reactions within protocellular environments has been proposed to be a decisive aspect to regulate chem. activity in space1. Today, cells and organisms adapt to signals2-6 by processing them through reaction networks that ultimately provide downstream functional responses and structural morphogenesis7,8. Re-enacting such signal processing in de novo-designed protocells is a profound challenge, but of high importance for understanding the design of adaptive systems with life-like traits. We report on engineered all-DNA protocells9 harbouring an artificial metalloenzyme10 whose olefin metathesis activity leads to downstream morphogenetic protocellular responses with varying levels of complexity. The artificial metalloenzyme catalyzes the uncaging of a pro-fluorescent signal mol. that generates a self-reporting fluorescent metabolite designed to weaken DNA duplex interactions. This leads to pronounced growth, intraparticular functional adaptation in the presence of a fluorescent DNA mechanosensor11 or interparticle protocell fusion. Such processes mimic chem. transduced processes found in cell adaptation and cell-to-cell adhesion. Our concept showcases new opportunities to study life-like behavior via abiotic bioorthogonal chem. and mech. transformations in synthetic protocells. Furthermore, it reveals a strategy for inducing complex behavior in adaptive and communicating soft-matter microsystems, and it illustrates how dynamic properties can be upregulated and sustained in micro-compartmentalized media.
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  18. 18
    Guindani, C.; da Silva, L. C.; Cao, S.; Ivanov, T.; Landfester, K. Synthetic Cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems. Angew. Chem., Int. Ed. 2022, 61, e202110855  DOI: 10.1002/anie.202110855
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    18
    Review on synthetic cells from simple bio-inspired modules to sophisticated integrated systems
    Guindani, Camila; da Silva, Lucas Caire; Cao, Shoupeng; Ivanov, Tsvetomir; Landfester, Katharina
    Angewandte Chemie, International Edition (2022), 61 (16), e202110855CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Bottom-up synthetic biol. is the science of building systems that mimic the structure and function of living cells from scratch. To do this, researchers combine tools from chem., materials science, and biochem. to develop functional and structural building blocks to construct synthetic cell-like systems. The many strategies and materials that have been developed in recent decades have enabled scientists to engineer synthetic cells and organelles that mimic the essential functions and behaviors of natural cells. Examples include synthetic cells that can synthesize their own ATP using light, maintain metabolic reactions through enzymic networks, perform gene replication, and even grow and divide. In this Review, we discuss recent developments in the design and construction of synthetic cells and organelles using the bottom-up approach. Our goal is to present representative synthetic cells of increasing complexity as well as strategies for solving distinct challenges in bottom-up synthetic biol.
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  19. 19
    Elani, Y. Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology. Angew. Chem., Int. Ed. 2021, 60, 5602– 5611,  DOI: 10.1002/anie.202006941
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    19
    Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology
    Elani, Yuval
    Angewandte Chemie, International Edition (2021), 60 (11), 5602-5611CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The construction of artificial cells from inanimate mol. building blocks is one of the grand challenges of our time. In addn. to being used as simplified cell models to decipher the rules of life, artificial cells have the potential to be designed as micromachines deployed in a host of clin. and industrial applications. The attractions of engineering artificial cells from scratch, as opposed to re-engineering living biol. cells, are varied. However, it is clear that artificial cells cannot currently match the power and behavioral sophistication of their biol. counterparts. Given this, many in the synthetic biol. community have started to ask: is it possible to interface biol. and artificial cells together to create hybrid living/synthetic systems that leverage the advantages of both. This article will discuss the motivation behind this cellular bionics approach, in which the boundaries between living and non-living matter are blurred by bridging top-down and bottom-up synthetic biol. It details the state of play of this nascent field and introduces three generalised hybridization modes that have emerged.
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  20. 20
    Sato, Y.; Sakamoto, T.; Takinoue, M. Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets. Sci. Adv. 2020, 6, eaba3471  DOI: 10.1126/sciadv.aba3471
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    Aufinger, L.; Simmel, F. C. Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions. Angew. Chem., Int. Ed. 2018, 57, 17245– 17248,  DOI: 10.1002/anie.201809374
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    21
    Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions
    Aufinger, Lukas; Simmel, Friedrich C.
    Angewandte Chemie, International Edition (2018), 57 (52), 17245-17248CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Gel-based artificial organelles have been developed that enable sequence-specific and programmable localization of cell-free transcription and translation reactions inside an artificial cellular system. To this end, the authors use agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. Also mRNA mols., produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles contg. their cognate DNA triggers. Spatial confinement of transcription and translation in sep. organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.
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  22. 22
    Sehgal, P. B.; Westley, J.; Lerea, K. M.; DiSenso-Browne, S.; Etlinger, J. D. Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs): Biomolecular condensates in cell biology and virology. Anal. Biochem. 2020, 597, 113691,  DOI: 10.1016/j.ab.2020.113691
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    22
    Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs)
    Sehgal, Pravin B.; Westley, Jenna; Lerea, Kenneth M.; DiSenso-Browne, Susan; Etlinger, Joseph D.
    Analytical Biochemistry (2020), 597 (), 113691CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    A review. Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-sepd. biomol. condensates are increasingly viewed as crit. in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often assoc. with low affinity; thus cell integrity can be crit. to the maintenance of the full complement of resp. MLO components. Phase-sepd. condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic "crowding" (as obsd. with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-sepn. properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurol. diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease). Thus, studies of liq.-like phase sepn. (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biol. and the pathogenesis of human diseases.
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    Song, D.; Jo, Y.; Choi, J.-M.; Jung, Y. Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions. Nat. Commun. 2020, 11, 5642,  DOI: 10.1038/s41467-020-19476-4
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    23
    Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions
    Song, Daesun; Jo, Yongsang; Choi, Jeong-Mo; Jung, Yongwon
    Nature Communications (2020), 11 (1), 5642CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liq.-liq. phase sepn., biomol. behaviors such as protein interactions inside these liq. compartments have been largely unexplored. Here we report quant. measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase sepn. conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase sepn. model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concns. inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase sepn. compartmentalization: (1) clients are selectively recruited into compartments, leading to concn. enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.
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  24. 24
    Trantidou, T.; Friddin, M.; Elani, Y.; Brooks, N. J.; Law, R. V.; Seddon, J. M.; Ces, O. Engineering Compartmentalized Biomimetic Micro- and Nanocontainers. ACS Nano 2017, 11, 6549– 6565,  DOI: 10.1021/acsnano.7b03245
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    24
    Engineering Compartmentalized Biomimetic Micro- and Nanocontainers
    Trantidou, Tatiana; Friddin, Mark; Elani, Yuval; Brooks, Nicholas J.; Law, Robert V.; Seddon, John M.; Ces, Oscar
    ACS Nano (2017), 11 (7), 6549-6565CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    A review. Compartmentalization of biol. content and function is a key architectural feature in biol., where membrane bound micro- and nanocompartments were used for performing a host of highly specialized and tightly regulated biol. functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, the authors discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches are discussed. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). The authors outline key examples of how such structures have been functionalized with biol. and synthetic machinery, for example, to manuf. and deliver drugs and metabolic compds., to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, the authors outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biol.
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  25. 25
    Seeman, N. C.; Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 2018, 3, 17068,  DOI: 10.1038/natrevmats.2017.68
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    25
    DNA nanotechnology
    Seeman, Nadrian C.; Sleiman, Hanadi F.
    Nature Reviews Materials (2018), 3 (1), 17068CODEN: NRMADL; ISSN:2058-8437. (Nature Research)
    DNA is the mol. that stores and transmits genetic information in biol. systems. The field of DNA nanotechnol. takes this mol. out of its biol. context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnol., and has been revolutionary in our ability to control mol. self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomol. structure detn., drug delivery and synthetic biol. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.
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    Rubio-Sánchez, R.; Fabrini, G.; Cicuta, P.; Di Michele, L. Amphiphilic DNA Nanostructures for Bottom-Up Synthetic Biology. Chem. Commun. 2021, 57, 12725– 12740,  DOI: 10.1039/D1CC04311K
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    26
    Amphiphilic DNA nanostructures for bottom-up synthetic biology
    Rubio-Sanchez, Roger; Fabrini, Giacomo; Cicuta, Pietro; Di Michele, Lorenzo
    Chemical Communications (Cambridge, United Kingdom) (2021), 57 (95), 12725-12740CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A review. DNA nanotechnol. enables the construction of sophisticated biomimetic nanomachines that are increasingly central to the growing efforts of creating complex cell-like entities from the bottom-up. DNA nanostructures have been proposed as both structural and functional elements of these artificial cells, and in many instances are decorated with hydrophobic moieties to enable interfacing with synthetic lipid bilayers or regulating bulk self-organization. In this feature article we review recent efforts to design biomimetic membrane-anchored DNA nanostructures capable of imparting complex functionalities to cell-like objects, such as regulated adhesion, tissue formation, communication and transport. We then discuss the ability of hydrophobic modifications to enable the self-assembly of DNA-based nanostructured frameworks with prescribed morphol. and functionality, and explore the relevance of these novel materials for artificial cell science and beyond. Finally, we comment on the yet mostly unexpressed potential of amphiphilic DNA-nanotechnol. as a complete toolbox for bottom-up synthetic biol. - a figurative and literal scaffold upon which the next generation of synthetic cells could be built.
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  27. 27
    Abe, K.; Kawamata, I.; Nomura, S. I. M.; Murata, S. Programmable reactions and diffusion using DNA for pattern formation in hydrogel medium. Mol. Syst. Des. Eng. 2019, 4, 639– 643,  DOI: 10.1039/C9ME00004F
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    27
    Programmable reactions and diffusion using DNA for pattern formation in hydrogel medium
    Abe, Keita; Kawamata, Ibuki; Nomura, Shin-ichiro M.; Murata, Satoshi
    Molecular Systems Design & Engineering (2019), 4 (3), 639-643CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)
    We demonstrate a method of pattern formation based on an artificial reaction diffusion system in hydrogel medium. By designing both the reaction term and the diffusion term of the system, we have succeeded in generating a sustainable DNA pattern in the gel. A DNA logic gate anchored in the gel detected the diffused mols. from distant source points to mark the equidistant region from the source points, which produced a Voronoi pattern in the gel. Combined with a diffusion modulation method for DNA mols., the Voronoi pattern was modified as a weighted Voronoi pattern with different morphologies. The proposed framework will be useful in designing a structured gel system responsive to mol. signals.
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    Chen, S.; Seelig, G. Programmable patterns in a DNA-based reaction-diffusion system. Soft Matter 2020, 16, 3555– 3563,  DOI: 10.1039/C9SM02413A
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    28
    Programmable patterns in a DNA-based reaction-diffusion system
    Chen, Sifang; Seelig, Georg
    Soft Matter (2020), 16 (14), 3555-3563CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
    Biol. offers compelling proof that macroscopic "living materials" can emerge from reactions between diffusing biomols. Here, we show that mol. self-organization could be a similarly powerful approach for engineering functional synthetic materials. We introduce a programmable DNA embedded hydrogel that produces tunable patterns at the centimeter length scale. We generate these patterns by implementing chem. reaction networks through synthetic DNA complexes, embedding the complexes in the hydrogel, and triggering with locally applied input DNA strands. We first demonstrate ring pattern formation around a circular input cavity and show that the ring width and intensity can be predictably tuned. Then, we create patterns of increasing complexity, including concentric rings and non-isotropic patterns. Finally, we show "destructive" and "constructive" interference patterns, by combining several ring-forming modules in the gel and triggering them from multiple sources. We further show that computer simulations based on the reaction-diffusion model can predict and inform the programming of target patterns.
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    Zadorin, A. S.; Rondelez, Y.; Gines, G.; Dilhas, V.; Urtel, G.; Zambrano, A.; Galas, J. C.; Estevez-Torres, A. Synthesis and materialization of a reaction-diffusion French flag pattern. Nat. Chem. 2017, 9, 990– 996,  DOI: 10.1038/nchem.2770
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    29
    Synthesis and materialization of a reaction-diffusion French flag pattern
    Zadorin, Anton S.; Rondelez, Yannick; Gines, Guillaume; Dilhas, Vadim; Urtel, Georg; Zambrano, Adrian; Galas, Jean-Christophe; Estevez-Torres, Andre
    Nature Chemistry (2017), 9 (10), 990-996CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    During embryo development, patterns of protein concn. appear in response to morphogen gradients. These patterns provide spatial and chem. information that directs the fate of the underlying cells. Here, the authors emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material. First, the authors use DNA-based reaction networks to synthesize a French flag, an archetypal pattern composed of three chem. distinct zones with sharp borders whose synthetic analog has remained elusive. A bistable network within a shallow concn. gradient creates an immobile, sharp and long-lasting concn. front through a reaction-diffusion mechanism. The combination of two bistable circuits generates a French flag pattern whose 'phenotype' can be reprogrammed by network mutation. Second, these concn. patterns control the macroscopic organization of DNA-decorated particles, inducing a French flag pattern of colloidal aggregation. This exptl. framework could be used to test reaction-diffusion models and fabricate soft materials following an autonomous developmental program.
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    Scalise, D.; Schulman, R. Designing modular reaction-diffusion programs for complex pattern formation. Technology 2014, 02, 55– 66,  DOI: 10.1142/S2339547814500071
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    Brady, R. A.; Brooks, N. J.; Cicuta, P.; Di Michele, L. Crystallization of Amphiphilic DNA C-Stars. Nano Lett. 2017, 17, 3276– 3281,  DOI: 10.1021/acs.nanolett.7b00980
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    31
    Crystallization of Amphiphilic DNA C-Stars
    Brady, Ryan A.; Brooks, Nicholas J.; Cicuta, Pietro; Di Michele, Lorenzo
    Nano Letters (2017), 17 (5), 3276-3281CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Many emerging technologies require materials with well-defined three-dimensional nanoscale architectures. Prodn. of these structures is currently underpinned by self-assembling amphiphilic macromols. or engineered all-DNA building blocks. Both of these approaches produce restricted ranges of crystal geometries due to synthetic amphiphiles' simple shape and limited specificity, or the tech. difficulties in designing space-filling DNA motifs with targeted shapes. The authors have overcome these limitations with amphiphilic DNA nanostructures, or "C-Stars", that combine the design freedom and facile functionalization of DNA-based materials with robust hydrophobic interactions. C-Stars self-assemble into single crystals exceeding 40 μm in size with lattice parameters exceeding 20 nm.
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    Brady, R. A.; Brooks, N. J.; Foderà, V.; Cicuta, P.; Di Michele, L. Amphiphilic-DNA Platform for the Design of Crystalline Frameworks with Programmable Structure and Functionality. J. Am. Chem. Soc. 2018, 140, 15384– 15392,  DOI: 10.1021/jacs.8b09143
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    32
    Amphiphilic-DNA Platform for the Design of Crystalline Frameworks with Programmable Structure and Functionality
    Brady, Ryan A.; Brooks, Nicholas J.; Fodera, Vito; Cicuta, Pietro; Di Michele, Lorenzo
    Journal of the American Chemical Society (2018), 140 (45), 15384-15392CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The reliable prepn. of functional, ordered, nanostructured frameworks would be a game changer for many emerging technologies, from energy storage to nanomedicine. Underpinned by the excellent mol. recognition of nucleic acids, along with their facile synthesis and breadth of available functionalizations, DNA nanotechnol. is widely acknowledged as a prime route for the rational design of nanostructured materials. Yet, the prepn. of cryst. DNA frameworks with programmable structure and functionality remains a challenge. Here we demonstrate the potential of simple amphiphilic DNA motifs, dubbed "C-stars", as a versatile platform for the design of programmable DNA crystals. In contrast to all-DNA materials, in which structure depends on the precise mol. details of individual building blocks, the self-assembly of C-stars is controlled uniquely by their topol. and symmetry. Exploiting this robust self-assembly principle, we design a range of topol. identical, but structurally and chem. distinct C-stars that following a one-pot reaction self-assemble into highly porous, functional, cryst. frameworks. Simple design variations allow us to fine-tune the lattice parameter and thus control the partitioning of macromols. within the frameworks, embed responsive motifs that can induce isothermal disassembly, and include chem. moieties to capture target proteins specifically and reversibly.
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    Brady, R. A.; Kaufhold, W. T.; Brooks, N. J.; Foderà, V.; Di Michele, L. Flexibility defines structure in crystals of amphiphilic DNA nanostars. J. Phys.: Condens. Matter 2019, 31, 074003,  DOI: 10.1088/1361-648X/aaf4a1
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    33
    Flexibility defines structure in crystals of amphiphilic DNA nanostars
    Brady, Ryan A.; Kaufhold, Will T.; Brooks, Nicholas J.; Fodera, Vito; Michele, Lorenzo Di
    Journal of Physics: Condensed Matter (2019), 31 (7), 074003/1-074003/11CODEN: JCOMEL; ISSN:0953-8984. (IOP Publishing Ltd.)
    DNA nanostructures with programmable shape and interactions can be used as building blocks for the self-assembly of cryst. materials with prescribed nanoscale features, holding a vast technol. potential. Structural rigidity and bond directionality have been recognized as key design features for DNA motifs to sustain long-range order in 3D, but the practical challenges assocd. with prescribing building-block geometry with sufficient accuracy have limited the variety of available designs. We have recently introduced a novel platform for the one-pot prepn. of cryst. DNA frameworks supported by a combination of Watson-Crick base pairing and hydrophobic forces. Here we use small angle x-ray scattering and coarse-grained mol. simulations to demonstrate that, as opposed to available all-DNA approaches, amphiphilic motifs do not rely on structural rigidity to support long-range order. Instead, the flexibility of amphiphilic DNA building-blocks is a crucial feature for successful crystn.
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  34. 34
    Fabrini, G.; Minard, A.; Brady, R. A.; Di Antonio, M.; Di Michele, L. Cation-Responsive and Photocleavable Hydrogels from Noncanonical Amphiphilic DNA Nanostructures. Nano Lett. 2022, 22, 602– 611,  DOI: 10.1021/acs.nanolett.1c03314
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    Cation-Responsive and Photocleavable Hydrogels from Noncanonical Amphiphilic DNA Nanostructures
    Fabrini, Giacomo; Minard, Aisling; Brady, Ryan A.; Di Antonio, Marco; Di Michele, Lorenzo
    Nano Letters (2022), 22 (2), 602-611CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Thanks to its biocompatibility, versatility, and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering, and drug delivery. Of particular importance for in vivo applications is the possibility of making such nanomaterials responsive to physiol. stimuli. Here, we demonstrate how combining noncanonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanostructures, which we termed "Quad-Stars", capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the hydrogel aggregates can be photo-disassembled upon near-UV irradn. in the presence of a porphyrin photosensitizer. The combined reversibility of assembly, responsiveness, and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.
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    Walczak, M.; Brady, R. A.; Mancini, L.; Contini, C.; Rubio-Sánchez, R.; Kaufhold, W. T.; Cicuta, P.; Di Michele, L. Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment. Nat. Commun. 2021, 12, 4743,  DOI: 10.1038/s41467-021-24989-7
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    Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment
    Walczak, Michal; Brady, Ryan A.; Mancini, Leonardo; Contini, Claudia; Rubio-Sanchez, Roger; Kaufhold, William T.; Cicuta, Pietro; Di Michele, Lorenzo
    Nature Communications (2021), 12 (1), 4743CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Biol. has evolved a variety of agents capable of permeabilizing and disrupting lipid membranes, from amyloid aggregates, to antimicrobial peptides, to venom compds. While often assocd. with disease or toxicity, these agents are also central to many biosensing and therapeutic technologies. Here, we introduce a class of synthetic, DNA-based particles capable of disrupting lipid membranes. The particles have finely programmable size, and self-assemble from all-DNA and cholesterol-DNA nanostructures, the latter forming a membrane-adhesive core and the former a protective hydrophilic corona. We show that the corona can be selectively displaced with a mol. cue, exposing the 'Sicky' core. Unprotected particles adhere to synthetic lipid vesicles, which in turn enhances membrane permeability and leads to vesicle collapse. Furthermore, particle-particle coalescence leads to the formation of gel-like DNA aggregates that envelop surviving vesicles. This response is reminiscent of pathogen immobilization through immune cells secretion of DNA networks, as we demonstrate by trapping E. coli bacteria.
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    Zhang, D. Y.; Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 2009, 131, 17303– 17314,  DOI: 10.1021/ja906987s
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    Control of DNA Strand Displacement Kinetics using Toehold Exchange
    Zhang, David Yu; Winfree, Erik
    Journal of the American Chemical Society (2009), 131 (47), 17303-17314CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA mols. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quant. predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.
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    Simmel, F. C.; Yurke, B.; Singh, H. R. Principles and Applications of Nucleic Acid Strand Displacement Reactions. Chem. Rev. 2019, 119, 6326– 6369,  DOI: 10.1021/acs.chemrev.8b00580
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    Principles and Applications of Nucleic Acid Strand Displacement Reactions
    Simmel, Friedrich C.; Yurke, Bernard; Singh, Hari R.
    Chemical Reviews (Washington, DC, United States) (2019), 119 (10), 6326-6369CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Dynamic DNA nanotechnol., a subfield of DNA nanotechnol., is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addn., the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNA-based nanomachine (Yurke, B.; et al. Nature 2000, 406, 605-608). Since then, toehold-mediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnol. has grown exponentially. Besides mol. machines, the field has produced enzyme-free catalytic systems, all DNA chem. oscillators and the most complex mol. computers yet devised. Enzyme-free catalytic systems can function as chem. amplifiers and as such have received considerable attention for sensing and detection applications in chem. and medical diagnostics. Strand-displacement reactions have been combined with other enzymically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol.2015, 11, 287-294). Strand-displacement principles have also been applied in synthetic biol. to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnol. over the past years, the field now seems poised for practical application.
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    Stellwagen, E.; Lu, Y.; Stellwagen, N. C. Unified description of electrophoresis and diffusion for DNA and other polyions. Biochemistry 2003, 42, 11745– 50,  DOI: 10.1021/bi035203p
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    Unified Description of Electrophoresis and Diffusion for DNA and Other Polyions
    Stellwagen, Earle; Lu, Yongjun; Stellwagen, Nancy C.
    Biochemistry (2003), 42 (40), 11745-11750CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The electrophoretic mobilities and diffusion coeffs. of single- and double-stranded DNA mols. up to 50,000 bases or base pairs in size have been analyzed, using mobilities and diffusion coeffs. either measured by capillary electrophoresis or taken from the literature. The Einstein equation suggests that the electrophoretic mobilities (μ) and diffusion coeffs. (D) should be related by the expression μ/D = Q/kBT, where Q is the charge of the polyion (Q = zeo, where z is the no. of charged residues and eo is the fundamental electronic charge), kB is Boltzmann's const., and T is the abs. temp. If this equation were true, the ratio μ/zD should be a const. equal to eo/kBT (39.6 V-1) at 20°. However, the ratio μ/zD decreases with an increase in mol. wt. for both single- and double-stranded DNAs. The mobilities and diffusion coeffs. are better described by the modified Einstein equation μ/NmD = eo/kBT, where N is the no. of repeat units (bases or base pairs) in the DNA and m is a const. equal to the power law dependence of the diffusion coeffs. on mol. wt. The av. value of the ratio μ/NmD is 40±4 V-1 for 36 single- and double-stranded DNA mols. of different sizes, close to the theor. expected value. The generality of the modified Einstein equation is demonstrated by analyzing literature values for sodium polystyrenesulfonate (PSS). The av. value of the ratio μ/NmD is 35±6 V-1 for 14 PSS samples contg. up to 855 monomers.
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    Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Diffusion of macromolecules in agarose gels: Comparison of linear and globular configurations. Biophys. J. 1999, 77, 542– 552,  DOI: 10.1016/S0006-3495(99)76911-0
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    Diffusion of macromolecules in agarose gels: Comparison of linear and globular configurations
    Pluen, Alain; Netti, Paolo A.; Jain, Rakesh K.; Berk, David A.
    Biophysical Journal (1999), 77 (1), 542-552CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The diffusion coeffs. (D) of different types of macromols. (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in soln. and in 2% agarose gels to compare transport properties of these macromols. Diffusion measurements were conducted with concns. low enough to avoid macromol. interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromols. were analyzed sep. As chain length increases, diffusion coeffs. of DNA show a clear shift from a Rouse-like behavior (DG ≃ N0-0.5) to a reptational behavior (DG ≃ N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS soln. was estd. Diffusion coeffs. of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estn. of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromols. exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.
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    Schuck, P. Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport. Biophys. J. 1996, 70, 1230– 1249,  DOI: 10.1016/S0006-3495(96)79681-9
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    Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport
    Schuck, Peter
    Biophysical Journal (1996), 70 (3), 1230-49CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The influence of mass transport on ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor, was investigated. A one-dimensional computer model for the mass transport of ligand between the bulk soln. and the polymer gel and within the gel was employed, and the influence of the diffusion coeff., the partition coeff., the thickness of the matrix, and the distribution of immobilized receptor were studied for a variety of conditions. Under conditions that may apply to many published exptl. studies, diffusion within the matrix was found to decrease the overall ligand transport significantly. For relatively slow reactions, small spatial gradients of free and bound ligand in the gel are found, whereas for relatively rapid reactions strong inhomogeneities of ligand within the gel occur before establishment of equil. Several types of deviations from ideal pseudo-first-order binding progress curves are described that resemble those of published exptl. data. Extremely transport limited reactions can in some cases be fitted with apparently ideal binding progress curves, although with apparent reaction rates that are much lower than the true reaction rates. Nevertheless, the ratio of the apparent rate consts. can be semiquant. consistent with the true equil. const. Apparently "cooperative" binding can result from high chem. on rates at high receptor satn. Dissocn. in the presence of transport limitation was found to be well described empirically by a single or a double exponential, with both apparent rate consts. considerably lower than the intrinsic chem. rate const. Transport limitations in the gel can introduce many generally unknown factors into the binding progress curve. The simulations suggest that unexpected deviations from ideal binding progress curves may be due to highly transport influenced binding kinetics. The use of a thinner polymer matrix could significantly increase the range of detectable rate consts.
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    Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions
    Semenov, Sergey N.; Markvoort, Albert J.; Gevers, Wouter B. L.; Piruska, Aigars; de Greef, Tom F. A.; Huck, Wilhelm T. S.
    Biophysical Journal (2013), 105 (4), 1057-1066CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Delineating design principles of biol. systems by reconstitution of purified components offers a platform to gauge the influence of crit. physicochem. parameters on minimal biol. systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films contg. immobilized substrates and reversible inhibitors. Quant. fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addn. of strong inhibitors, consistent with a math. model of mol. titrn. The results confirm that ultrasensitive and threshold effects at the mol. level can convert a graded input signal to a steep spatial response at macroscopic length scales.
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    Raue, A.; Kreutz, C.; Maiwald, T.; Bachmann, J.; Schilling, M.; Klingmüller, U.; Timmer, J. Structural and practical identifiability analysis of partially observed dynamical models by exploiting the profile likelihood. Bioinformatics 2009, 25, 1923– 1929,  DOI: 10.1093/bioinformatics/btp358
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    Structural and practical identifiability analysis of partially observed dynamical models by exploiting the profile likelihood
    Raue, A.; Kreutz, C.; Maiwald, T.; Bachmann, J.; Schilling, M.; Klingmueller, U.; Timmer, J.
    Bioinformatics (2009), 25 (15), 1923-1929CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
    Math. description of biol. reaction networks by differential equations leads to large models whose parameters are calibrated to optimally explain exptl. data. Often only parts of the model can be obsd. directly. Given a model that sufficiently describes the measured data, it is important to infer how well model parameters are detd. by the amt. and quality of exptl. data. This knowledge is essential for further investigation of model predictions. For this reason a major topic in modeling is identifiability anal. The authors suggest an approach that exploits the profile likelihood. It enables to detect structural non-identifiabilities, which manifest in functionally related model parameters. Furthermore, practical non-identifiabilities are detected, that might arise due to limited amt. and quality of exptl. data. Last but not least confidence intervals can be derived. The results are easy to interpret and can be used for exptl. planning and for model redn. Availability: An implementation is freely available for MATLAB and the PottersWheel modeling toolbox at http://web.me.com/andreas.raue/profile/software.html. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.
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    Kar, S.; Ellington, A. D. Construction of synthetic T7 RNA polymerase expression systems. Methods 2018, 143, 110– 120,  DOI: 10.1016/j.ymeth.2018.02.022
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    Construction of synthetic T7 RNA polymerase expression systems
    Kar, Shaunak; Ellington, Andrew D.
    Methods (Amsterdam, Netherlands) (2018), 143 (), 110-120CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
    T7 RNA polymerase (T7 RNAP) is one of the preferred workhorses for recombinant gene expression, owing in part to its high transcriptional activity and the fact that it has a small (17 base-pair), easily manipulated promoter. However, the high activity of the enzyme also often leads to an increased fitness burden on the host, limiting the predictability of its interactions and impact on host physiol., and potentially leading to mutations in constructs. Here we use a synthetic biol. approach to design and characterize a panel of T7 RNAP expression circuits with different modes of regulation that enable the reliable expression of downstream targets under a variety of conditions. First, we describe the construction of a minimal T7 RNAP expression system that is inducible by a small mol. anhydrotetracycline (aTc), and then characterize a self-limiting T7 RNAP expression circuit that provides better control over the amt. of T7 RNAP produced upon induction. Finally, we characterize a so-called T7 RNAP homeostasis circuit that leads to constitutive, continuous, and sub-toxic levels of T7 RNAP. Coupled with previously characterized mutant T7 RNAP promoters in vitro, we demonstrate that this modular framework can be used to achieve precise and predictable levels of output (sfGFP) in vivo. This new framework should now allow modeling and construction of T7 RNAP expression constructs that expand the utility of this enzyme for driving a variety of synthetic circuits and constructs.
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    Filonov, G. S.; Moon, J. D.; Svensen, N.; Jaffrey, S. R. Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution. J. Am. Chem. Soc. 2014, 136, 16299– 16308,  DOI: 10.1021/ja508478x
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    Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution
    Filonov, Grigory S.; Moon, Jared D.; Svensen, Nina; Jaffrey, Samie R.
    Journal of the American Chemical Society (2014), 136 (46), 16299-16308CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small mols. in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although addnl. highly fluorescent RNA-fluorophore complexes would extend the utility of this technol., the identification of novel RNA-fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Addnl., aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here the authors describe a platform for rapid generation of highly fluorescent RNA-fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, the authors identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concns. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.
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    Dana, H.; Chalbatani, G. M.; Mahmoodzadeh, H.; Karimloo, R.; Rezaiean, O.; Moradzadeh, A.; Mehmandoost, N.; Moazzen, F.; Mazraeh, A.; Marmari, V.; Ebrahimi, M.; Rashno, M. M.; Abadi, S. J.; Gharagouzlo, E. Molecular Mechanisms and Biological Functions of siRNA. Int. J. Biomed. Sci. 2017, 13 (2), 48– 57
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    Molecular Mechanisms and Biological Functions of siRNA
    Dana Hassan; Rezaiean Omid; Moradzadeh Amirreza; Mazraeh Ali; Marmari Vahid; Rashno Mohammad Menati; Chalbatani Ghanbar Mahmoodi; Mahmoodzadeh Habibollah; Gharagouzlo Elahe; Karimloo Rezvan; Mehmandoost Narges; Moazzen Fateme; Ebrahimi Mohammad; Abadi Saeid Jan
    International journal of biomedical science : IJBS (2017), 13 (2), 48-57 ISSN:1550-9702.
    One of the most important advances in biology has been the discovery that siRNA (small interfering RNA) is able to regulate the expression of genes, by a phenomenon known as RNAi (RNA interference). The discovery of RNAi, first in plants and Caenorhabditis elegans and later in mammalian cells, led to the emergence of a transformative view in biomedical research. siRNA has gained attention as a potential therapeutic reagent due to its ability to inhibit specific genes in many genetic diseases. siRNAs can be used as tools to study single gene function both in vivo and in-vitro and are an attractive new class of therapeutics, especially against undruggable targets for the treatment of cancer and other diseases. The siRNA delivery systems are categorized as non-viral and viral delivery systems. The non-viral delivery system includes polymers; Lipids; peptides etc. are the widely studied delivery systems for siRNA. Effective pharmacological use of siRNA requires 'carriers' that can deliver the siRNA to its intended site of action. The carriers assemble the siRNA into supramolecular complexes that display functional properties during the delivery process.
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    Moreno-Risueno, M. A.; Benfey, P. N. Time-based patterning in development: The role of oscillating gene expression. Transcription 2011, 2, 124– 129,  DOI: 10.4161/trns.2.3.15637
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    Time-based patterning in development: The role of oscillating gene expression
    Moreno-Risueno Miguel A; Benfey Philip N
    Transcription (2011), 2 (3), 124-129 ISSN:.
    Oscillating gene expression is a mechanism of patterning during development in both plants and animals. In vertebrates, oscillating gene expression establishes the musculoskeletal precursors (somites), while in plant roots it establishes the position of future organs (lateral roots). Both mechanisms constitute a specialized type of biological clock that converts temporal information into precise spatial patterns. Similarities, differences, and their functionality in organisms that evolved independently are discussed.
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    Figure 1. Reaction–diffusion patterning of amphiphilic DNA condensates. (a) Reaction–diffusion processes are used to pattern initially uniform DNA condensates and construct DNA-based artificial cells featuring distinct internal environments of controllable number and molecular makeup, unlocking the spatial engineering of functionality. (b) Foundational building block of the condensates, consisting of a locked four-way DNA junction with cholesterol moieties at the end of each arm. The constructs are composed of four distinct strands that form the junction (blue) and four identical cholesterolized oligonucleotides (orange). (31−33) One arm features an additional overhang connected to a base strand (b), which serves as a binding site for complementary freely diffusing patterning strands. The latter range between 16  (p1) and 40 nt (p8) in length and compete over (color-coded) overlapping binding domains on the base strand. p1 and p8 shown here are functionalized with Alexa 594 (red) and Alexa 488 (green) fluorophores, respectively. The stop strand (s) has the same sequence as the base strand and can be added in solution to sequester the excess patterning strands. Sequences of all the DNA oligonucleotides are provided in Table S1. (c) Assembly process for amphiphilic DNA condensates. Samples containing all single-stranded DNA components are slowly annealed from 90 to 20 °C, leading to the formation of a nanoporous framework in which the branched DNA motifs connect micelle-like hydrophobic cores where the cholesterol modifications localize, as previously reported. (31−33) Sample preparation details are provided in the Experimental Methods (SI). (d) Schematic depiction of the designed reaction–diffusion pathway. At time t = 0, condensates are exposed to a solution of p1 (short, red) and p8 (long, green) patterning strands in excess concentrations compared to the base strands. Short p1 DNA strands are able to diffuse inside the condensates faster than long p8 strands, allowing for prior binding to the base strand (red box). At later times, p8 strands then diffuse within the condensates and, due to the sequence design, are able to displace p1 strands via toeholding (36,37) (green box). The result is a sequence of two fronts that propagate inward through the condensate. (e) Series of confocal micrographs of the process discussed in panel c, where propagating fronts are visualized thanks to fluorescent modifications of p1 and p8. the scale bar represents 15 μm.

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    Figure 2

    Figure 2. Condensate patterning is predictable and customizable. (a–d) Patterning-strand scheme (-i) and equatorial confocal microscopy sections (-ii) for condensates patterned to form an increasing number of concentric domains, from one in system a to five in system d. Some patterning strands are fluorescently labeled with Alexa 594 (p1) and Alexa 488 (p5 and p8) while others do not bear modifications, resulting in dark regions intermitting the fluorescent shells in the confocal data. See Table S1 for the DNA sequences. The spatiotemporal evolution of the domain structure is visualized as the azimuthally averaged, normalized radial intensity profile I(r, t), where r is the radial coordinate defined from the centroid of the condensate and t is the time elapsed from exposure of the condensates to the patterning strands (-iii). For systems a–c, I(r, t) is compared with the fitted outcome of a reaction–diffusion numerical model (-iv). Note that early times are not shown in experimental color maps (gray bands) due to a delay between the time at which condensates were exposed to the patterning strand (t = 0) and the start of the confocal recording. See the Experimental Methods (SI) for information on image analysis and numerical modeling. For system d, subpanel d-iv shows the radial intensity profiles extracted from confocal images at t = 7 min, highlighting the presence of five distinct domains. The green dotted and red dashed lines mark the signals from the Alexa 488 (p5 and p8) and Alexa 594 (p1) channels, respectively, while the black solid line represents the overall intensity. All profiles are normalized by their highest value. (e) Domain propagation can be arrested by adding an excess of the stop strand (s) in solution (e-i, see also Figure 1a), as demonstrated in e-ii with confocal data for a system with three patterning strands (p1, p6, and p8). The stop strand was added at t = 16 min, after which no further pattern evolution was observed (besides photobleaching). Videos S1–S8 show the pattern evolution in individual condensates (even numbered) and larger fields of view (odd numbered). See the supplementary videos key in the SI. Scale bars represent 15 μm.

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    Figure 3

    Figure 3. Model fitting enables the extraction of reaction–diffusion parameters. (a) Schematic representation of the color parameters, which consist of an entry rate kin, a diffusion constant D, and a binding or displacement rate kon (top). With kon, we indicate both the second-order binding rate of a patterning strand to a free binding site and that of the toehold-mediated strand displacement process through which a longer patterning strand replaces a shorter one that previously occupied a binding site (bottom, see the Modeling Methods (SI). (b and c) Diffusion coefficients for the 40 nt patterning strand p8 and the 16 nt patterning strand p1, respectively. (d and e) Binding rates for the 40 nt patterning strand p8 and the 16 nt patterning strand p1, respectively. Data are shown for samples with one patterning strand (p1 or p8; Figures 2a, S3, and S4; N = 33 condensates for p1 and N = 29 condensates for p8), two patterning strands (p1 and p8; Figures 2b and S5; N = 23 condensates) and three patterning strands (p1, p6, and p8; Figures 2c and S6; N = 43 condensates). The results are displayed as box plots with highlighted median, upper, and lower quartiles (box); 50th centile (whiskers) outliers are excluded. Overlaid on the box plots are the means (symbol) and standard deviations (error bar the same color as the symbol) of the distributions.

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    Figure 4

    Figure 4. Spatially distributed functionality in a model artificial cell. (a) Schematics of the functional nucleic acid machinery in the nucleus (cyan) and shell regions (orange). In the nucleus, connected to the base strand are a bridge (r) strand and the template (t) strand. Together, these form a double-stranded T7 promoter (pink) and a single-stranded polymerase template (purple, red) from which a polymerase (black) is able to synthesize Broccoli RNA aptamers (folded purple and red). These aptamers then form a complex with DFHBI molecules to become fluorescent (orange). The base strands in the shell region are connected to capture strands (c) with single-stranded overhangs (red) complementary to a free domain on the broccoli aptamer. Complementary DNA and RNA domains are shown in the same color. Protocols for patterning the ACs are detailed in the Experimental Methods (SI). (b) Mode of operation of the AC. The polymerase is added in solution alongside NTPs, DFHBI, and other components required for Broccoli synthesis, which diffuse through the shell (1) to reach the nucleus, where the aptamers are produced (2). The aptamers then diffuse outward and bind to the dedicated sites in the shell (3). (c) A series of confocal images (top) of an AC progressively building the Broccoli aptamer in the shell (orange). Note how the signal accumulates from the nucleus–shell interface and propagates outward. The nucleus is shown in cyan and progressively photobleaches. The dashed lines mark the physical boundary of the AC and that between the nucleus and the shell. The bottom images of bright-field images of the same AC overlaid onto (faint) confocal data, demonstrating that no physical change to the AC occurs during Broccoli synthesis. The reaction is initiated at time t = 0, as discussed in the Experimental Methods (SI). (d) Color map showing the evolution of the radial fluorescent intensity of the aptamer. The slope of the fluorescent front signals accumulation from the inside out, as highlighted by the white arrow. Dashed lines mark the nucleus–shell and shell/–olution boundaries. Videos S13–S16 show the responses of multiple ACs with different shell–nucleus size ratios. The scale bar represents 15 μm.

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      The cell cytosol is crowded with high concns. of many different biomacromols., which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biol. relevant macromols. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodol. This versatile approach for programmed spatial organization of biol. relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.
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    15. 15
      Simon, J. R.; Carroll, N. J.; Rubinstein, M.; Chilkoti, A.; López, G. P. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity. Nat. Chem. 2017, 9, 509– 515,  DOI: 10.1038/nchem.2715
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      15
      Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity
      Simon, Joseph R.; Carroll, Nick J.; Rubinstein, Michael; Chilkoti, Ashutosh; Lopez, Gabriel P.
      Nature Chemistry (2017), 9 (6), 509-515CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Dynamic protein-rich intracellular structures that contain phase-sepd. intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase sep. into diverse assemblies within droplet microenvironments. Using theor. analyses, we interpret the phase behavior of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-sepd. granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-mol.-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biol. and (3) mol.-level engineering of self-assembled recombinant IDP-rich materials.
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    16. 16
      Qiao, Y.; Li, M.; Booth, R.; Mann, S. Predatory behaviour in synthetic protocell communities. Nat. Chem. 2017, 9, 110– 119,  DOI: 10.1038/nchem.2617
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      16
      Predatory behaviour in synthetic protocell communities
      Qiao, Yan; Li, Mei; Booth, Richard; Mann, Stephen
      Nature Chemistry (2017), 9 (2), 110-119CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Recent progress in the chem. construction of colloidal objects comprising integrated biomimetic functions is paving the way towards rudimentary forms of artificial cell-like entities (protocells). Although several new types of protocells are currently available, the design of synthetic protocell communities and investigation of their collective behavior has received little attention. Here we demonstrate an artificial form of predatory behavior in a community of protease-contg. coacervate microdroplets and protein-polymer microcapsules (proteinosomes) that interact via electrostatic binding. The coacervate microdroplets act as killer protocells for the obliteration of the target proteinosome population by protease-induced lysis of the protein-polymer membrane. As a consequence, the proteinosome payload (dextran, single-stranded DNA, platinum nanoparticles) is trafficked into the attached coacervate microdroplets, which are then released as functionally modified killer protocells capable of rekilling. Our results highlight opportunities for the development of interacting artificial protocell communities, and provide a strategy for inducing collective behavior in soft matter microcompartmentalized systems and synthetic protocell consortia.
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    17. 17
      Samanta, A.; Sabatino, V.; Ward, T. R.; Walther, A. Functional and morphological adaptation in DNA protocells via signal processing prompted by artificial metalloenzymes. Nat. Nanotechnol. 2020, 15, 914– 921,  DOI: 10.1038/s41565-020-0761-y
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      17
      Functional and morphological adaptation in DNA protocells via signal processing prompted by artificial metalloenzymes
      Samanta, Avik; Sabatino, Valerio; Ward, Thomas R.; Walther, Andreas
      Nature Nanotechnology (2020), 15 (11), 914-921CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
      Abstr.: For life to emerge, the confinement of catalytic reactions within protocellular environments has been proposed to be a decisive aspect to regulate chem. activity in space1. Today, cells and organisms adapt to signals2-6 by processing them through reaction networks that ultimately provide downstream functional responses and structural morphogenesis7,8. Re-enacting such signal processing in de novo-designed protocells is a profound challenge, but of high importance for understanding the design of adaptive systems with life-like traits. We report on engineered all-DNA protocells9 harbouring an artificial metalloenzyme10 whose olefin metathesis activity leads to downstream morphogenetic protocellular responses with varying levels of complexity. The artificial metalloenzyme catalyzes the uncaging of a pro-fluorescent signal mol. that generates a self-reporting fluorescent metabolite designed to weaken DNA duplex interactions. This leads to pronounced growth, intraparticular functional adaptation in the presence of a fluorescent DNA mechanosensor11 or interparticle protocell fusion. Such processes mimic chem. transduced processes found in cell adaptation and cell-to-cell adhesion. Our concept showcases new opportunities to study life-like behavior via abiotic bioorthogonal chem. and mech. transformations in synthetic protocells. Furthermore, it reveals a strategy for inducing complex behavior in adaptive and communicating soft-matter microsystems, and it illustrates how dynamic properties can be upregulated and sustained in micro-compartmentalized media.
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    18. 18
      Guindani, C.; da Silva, L. C.; Cao, S.; Ivanov, T.; Landfester, K. Synthetic Cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems. Angew. Chem., Int. Ed. 2022, 61, e202110855  DOI: 10.1002/anie.202110855
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      18
      Review on synthetic cells from simple bio-inspired modules to sophisticated integrated systems
      Guindani, Camila; da Silva, Lucas Caire; Cao, Shoupeng; Ivanov, Tsvetomir; Landfester, Katharina
      Angewandte Chemie, International Edition (2022), 61 (16), e202110855CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Bottom-up synthetic biol. is the science of building systems that mimic the structure and function of living cells from scratch. To do this, researchers combine tools from chem., materials science, and biochem. to develop functional and structural building blocks to construct synthetic cell-like systems. The many strategies and materials that have been developed in recent decades have enabled scientists to engineer synthetic cells and organelles that mimic the essential functions and behaviors of natural cells. Examples include synthetic cells that can synthesize their own ATP using light, maintain metabolic reactions through enzymic networks, perform gene replication, and even grow and divide. In this Review, we discuss recent developments in the design and construction of synthetic cells and organelles using the bottom-up approach. Our goal is to present representative synthetic cells of increasing complexity as well as strategies for solving distinct challenges in bottom-up synthetic biol.
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    19. 19
      Elani, Y. Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology. Angew. Chem., Int. Ed. 2021, 60, 5602– 5611,  DOI: 10.1002/anie.202006941
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      19
      Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology
      Elani, Yuval
      Angewandte Chemie, International Edition (2021), 60 (11), 5602-5611CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The construction of artificial cells from inanimate mol. building blocks is one of the grand challenges of our time. In addn. to being used as simplified cell models to decipher the rules of life, artificial cells have the potential to be designed as micromachines deployed in a host of clin. and industrial applications. The attractions of engineering artificial cells from scratch, as opposed to re-engineering living biol. cells, are varied. However, it is clear that artificial cells cannot currently match the power and behavioral sophistication of their biol. counterparts. Given this, many in the synthetic biol. community have started to ask: is it possible to interface biol. and artificial cells together to create hybrid living/synthetic systems that leverage the advantages of both. This article will discuss the motivation behind this cellular bionics approach, in which the boundaries between living and non-living matter are blurred by bridging top-down and bottom-up synthetic biol. It details the state of play of this nascent field and introduces three generalised hybridization modes that have emerged.
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    20. 20
      Sato, Y.; Sakamoto, T.; Takinoue, M. Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets. Sci. Adv. 2020, 6, eaba3471  DOI: 10.1126/sciadv.aba3471
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    21. 21
      Aufinger, L.; Simmel, F. C. Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions. Angew. Chem., Int. Ed. 2018, 57, 17245– 17248,  DOI: 10.1002/anie.201809374
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      21
      Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions
      Aufinger, Lukas; Simmel, Friedrich C.
      Angewandte Chemie, International Edition (2018), 57 (52), 17245-17248CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Gel-based artificial organelles have been developed that enable sequence-specific and programmable localization of cell-free transcription and translation reactions inside an artificial cellular system. To this end, the authors use agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. Also mRNA mols., produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles contg. their cognate DNA triggers. Spatial confinement of transcription and translation in sep. organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.
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    22. 22
      Sehgal, P. B.; Westley, J.; Lerea, K. M.; DiSenso-Browne, S.; Etlinger, J. D. Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs): Biomolecular condensates in cell biology and virology. Anal. Biochem. 2020, 597, 113691,  DOI: 10.1016/j.ab.2020.113691
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      22
      Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs)
      Sehgal, Pravin B.; Westley, Jenna; Lerea, Kenneth M.; DiSenso-Browne, Susan; Etlinger, Joseph D.
      Analytical Biochemistry (2020), 597 (), 113691CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      A review. Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-sepd. biomol. condensates are increasingly viewed as crit. in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often assoc. with low affinity; thus cell integrity can be crit. to the maintenance of the full complement of resp. MLO components. Phase-sepd. condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic "crowding" (as obsd. with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-sepn. properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurol. diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease). Thus, studies of liq.-like phase sepn. (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biol. and the pathogenesis of human diseases.
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    23. 23
      Song, D.; Jo, Y.; Choi, J.-M.; Jung, Y. Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions. Nat. Commun. 2020, 11, 5642,  DOI: 10.1038/s41467-020-19476-4
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      23
      Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions
      Song, Daesun; Jo, Yongsang; Choi, Jeong-Mo; Jung, Yongwon
      Nature Communications (2020), 11 (1), 5642CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liq.-liq. phase sepn., biomol. behaviors such as protein interactions inside these liq. compartments have been largely unexplored. Here we report quant. measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase sepn. conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase sepn. model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concns. inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase sepn. compartmentalization: (1) clients are selectively recruited into compartments, leading to concn. enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.
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    24. 24
      Trantidou, T.; Friddin, M.; Elani, Y.; Brooks, N. J.; Law, R. V.; Seddon, J. M.; Ces, O. Engineering Compartmentalized Biomimetic Micro- and Nanocontainers. ACS Nano 2017, 11, 6549– 6565,  DOI: 10.1021/acsnano.7b03245
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      24
      Engineering Compartmentalized Biomimetic Micro- and Nanocontainers
      Trantidou, Tatiana; Friddin, Mark; Elani, Yuval; Brooks, Nicholas J.; Law, Robert V.; Seddon, John M.; Ces, Oscar
      ACS Nano (2017), 11 (7), 6549-6565CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      A review. Compartmentalization of biol. content and function is a key architectural feature in biol., where membrane bound micro- and nanocompartments were used for performing a host of highly specialized and tightly regulated biol. functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, the authors discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches are discussed. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). The authors outline key examples of how such structures have been functionalized with biol. and synthetic machinery, for example, to manuf. and deliver drugs and metabolic compds., to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, the authors outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biol.
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    25. 25
      Seeman, N. C.; Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 2018, 3, 17068,  DOI: 10.1038/natrevmats.2017.68
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      25
      DNA nanotechnology
      Seeman, Nadrian C.; Sleiman, Hanadi F.
      Nature Reviews Materials (2018), 3 (1), 17068CODEN: NRMADL; ISSN:2058-8437. (Nature Research)
      DNA is the mol. that stores and transmits genetic information in biol. systems. The field of DNA nanotechnol. takes this mol. out of its biol. context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnol., and has been revolutionary in our ability to control mol. self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomol. structure detn., drug delivery and synthetic biol. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.
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    26. 26
      Rubio-Sánchez, R.; Fabrini, G.; Cicuta, P.; Di Michele, L. Amphiphilic DNA Nanostructures for Bottom-Up Synthetic Biology. Chem. Commun. 2021, 57, 12725– 12740,  DOI: 10.1039/D1CC04311K
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      26
      Amphiphilic DNA nanostructures for bottom-up synthetic biology
      Rubio-Sanchez, Roger; Fabrini, Giacomo; Cicuta, Pietro; Di Michele, Lorenzo
      Chemical Communications (Cambridge, United Kingdom) (2021), 57 (95), 12725-12740CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A review. DNA nanotechnol. enables the construction of sophisticated biomimetic nanomachines that are increasingly central to the growing efforts of creating complex cell-like entities from the bottom-up. DNA nanostructures have been proposed as both structural and functional elements of these artificial cells, and in many instances are decorated with hydrophobic moieties to enable interfacing with synthetic lipid bilayers or regulating bulk self-organization. In this feature article we review recent efforts to design biomimetic membrane-anchored DNA nanostructures capable of imparting complex functionalities to cell-like objects, such as regulated adhesion, tissue formation, communication and transport. We then discuss the ability of hydrophobic modifications to enable the self-assembly of DNA-based nanostructured frameworks with prescribed morphol. and functionality, and explore the relevance of these novel materials for artificial cell science and beyond. Finally, we comment on the yet mostly unexpressed potential of amphiphilic DNA-nanotechnol. as a complete toolbox for bottom-up synthetic biol. - a figurative and literal scaffold upon which the next generation of synthetic cells could be built.
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    27. 27
      Abe, K.; Kawamata, I.; Nomura, S. I. M.; Murata, S. Programmable reactions and diffusion using DNA for pattern formation in hydrogel medium. Mol. Syst. Des. Eng. 2019, 4, 639– 643,  DOI: 10.1039/C9ME00004F
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      27
      Programmable reactions and diffusion using DNA for pattern formation in hydrogel medium
      Abe, Keita; Kawamata, Ibuki; Nomura, Shin-ichiro M.; Murata, Satoshi
      Molecular Systems Design & Engineering (2019), 4 (3), 639-643CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)
      We demonstrate a method of pattern formation based on an artificial reaction diffusion system in hydrogel medium. By designing both the reaction term and the diffusion term of the system, we have succeeded in generating a sustainable DNA pattern in the gel. A DNA logic gate anchored in the gel detected the diffused mols. from distant source points to mark the equidistant region from the source points, which produced a Voronoi pattern in the gel. Combined with a diffusion modulation method for DNA mols., the Voronoi pattern was modified as a weighted Voronoi pattern with different morphologies. The proposed framework will be useful in designing a structured gel system responsive to mol. signals.
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    28. 28
      Chen, S.; Seelig, G. Programmable patterns in a DNA-based reaction-diffusion system. Soft Matter 2020, 16, 3555– 3563,  DOI: 10.1039/C9SM02413A
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      28
      Programmable patterns in a DNA-based reaction-diffusion system
      Chen, Sifang; Seelig, Georg
      Soft Matter (2020), 16 (14), 3555-3563CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
      Biol. offers compelling proof that macroscopic "living materials" can emerge from reactions between diffusing biomols. Here, we show that mol. self-organization could be a similarly powerful approach for engineering functional synthetic materials. We introduce a programmable DNA embedded hydrogel that produces tunable patterns at the centimeter length scale. We generate these patterns by implementing chem. reaction networks through synthetic DNA complexes, embedding the complexes in the hydrogel, and triggering with locally applied input DNA strands. We first demonstrate ring pattern formation around a circular input cavity and show that the ring width and intensity can be predictably tuned. Then, we create patterns of increasing complexity, including concentric rings and non-isotropic patterns. Finally, we show "destructive" and "constructive" interference patterns, by combining several ring-forming modules in the gel and triggering them from multiple sources. We further show that computer simulations based on the reaction-diffusion model can predict and inform the programming of target patterns.
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    29. 29
      Zadorin, A. S.; Rondelez, Y.; Gines, G.; Dilhas, V.; Urtel, G.; Zambrano, A.; Galas, J. C.; Estevez-Torres, A. Synthesis and materialization of a reaction-diffusion French flag pattern. Nat. Chem. 2017, 9, 990– 996,  DOI: 10.1038/nchem.2770
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      29
      Synthesis and materialization of a reaction-diffusion French flag pattern
      Zadorin, Anton S.; Rondelez, Yannick; Gines, Guillaume; Dilhas, Vadim; Urtel, Georg; Zambrano, Adrian; Galas, Jean-Christophe; Estevez-Torres, Andre
      Nature Chemistry (2017), 9 (10), 990-996CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      During embryo development, patterns of protein concn. appear in response to morphogen gradients. These patterns provide spatial and chem. information that directs the fate of the underlying cells. Here, the authors emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material. First, the authors use DNA-based reaction networks to synthesize a French flag, an archetypal pattern composed of three chem. distinct zones with sharp borders whose synthetic analog has remained elusive. A bistable network within a shallow concn. gradient creates an immobile, sharp and long-lasting concn. front through a reaction-diffusion mechanism. The combination of two bistable circuits generates a French flag pattern whose 'phenotype' can be reprogrammed by network mutation. Second, these concn. patterns control the macroscopic organization of DNA-decorated particles, inducing a French flag pattern of colloidal aggregation. This exptl. framework could be used to test reaction-diffusion models and fabricate soft materials following an autonomous developmental program.
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    30. 30
      Scalise, D.; Schulman, R. Designing modular reaction-diffusion programs for complex pattern formation. Technology 2014, 02, 55– 66,  DOI: 10.1142/S2339547814500071
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      Brady, R. A.; Brooks, N. J.; Cicuta, P.; Di Michele, L. Crystallization of Amphiphilic DNA C-Stars. Nano Lett. 2017, 17, 3276– 3281,  DOI: 10.1021/acs.nanolett.7b00980
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      Crystallization of Amphiphilic DNA C-Stars
      Brady, Ryan A.; Brooks, Nicholas J.; Cicuta, Pietro; Di Michele, Lorenzo
      Nano Letters (2017), 17 (5), 3276-3281CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Many emerging technologies require materials with well-defined three-dimensional nanoscale architectures. Prodn. of these structures is currently underpinned by self-assembling amphiphilic macromols. or engineered all-DNA building blocks. Both of these approaches produce restricted ranges of crystal geometries due to synthetic amphiphiles' simple shape and limited specificity, or the tech. difficulties in designing space-filling DNA motifs with targeted shapes. The authors have overcome these limitations with amphiphilic DNA nanostructures, or "C-Stars", that combine the design freedom and facile functionalization of DNA-based materials with robust hydrophobic interactions. C-Stars self-assemble into single crystals exceeding 40 μm in size with lattice parameters exceeding 20 nm.
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      Brady, R. A.; Brooks, N. J.; Foderà, V.; Cicuta, P.; Di Michele, L. Amphiphilic-DNA Platform for the Design of Crystalline Frameworks with Programmable Structure and Functionality. J. Am. Chem. Soc. 2018, 140, 15384– 15392,  DOI: 10.1021/jacs.8b09143
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      32
      Amphiphilic-DNA Platform for the Design of Crystalline Frameworks with Programmable Structure and Functionality
      Brady, Ryan A.; Brooks, Nicholas J.; Fodera, Vito; Cicuta, Pietro; Di Michele, Lorenzo
      Journal of the American Chemical Society (2018), 140 (45), 15384-15392CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The reliable prepn. of functional, ordered, nanostructured frameworks would be a game changer for many emerging technologies, from energy storage to nanomedicine. Underpinned by the excellent mol. recognition of nucleic acids, along with their facile synthesis and breadth of available functionalizations, DNA nanotechnol. is widely acknowledged as a prime route for the rational design of nanostructured materials. Yet, the prepn. of cryst. DNA frameworks with programmable structure and functionality remains a challenge. Here we demonstrate the potential of simple amphiphilic DNA motifs, dubbed "C-stars", as a versatile platform for the design of programmable DNA crystals. In contrast to all-DNA materials, in which structure depends on the precise mol. details of individual building blocks, the self-assembly of C-stars is controlled uniquely by their topol. and symmetry. Exploiting this robust self-assembly principle, we design a range of topol. identical, but structurally and chem. distinct C-stars that following a one-pot reaction self-assemble into highly porous, functional, cryst. frameworks. Simple design variations allow us to fine-tune the lattice parameter and thus control the partitioning of macromols. within the frameworks, embed responsive motifs that can induce isothermal disassembly, and include chem. moieties to capture target proteins specifically and reversibly.
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    33. 33
      Brady, R. A.; Kaufhold, W. T.; Brooks, N. J.; Foderà, V.; Di Michele, L. Flexibility defines structure in crystals of amphiphilic DNA nanostars. J. Phys.: Condens. Matter 2019, 31, 074003,  DOI: 10.1088/1361-648X/aaf4a1
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      33
      Flexibility defines structure in crystals of amphiphilic DNA nanostars
      Brady, Ryan A.; Kaufhold, Will T.; Brooks, Nicholas J.; Fodera, Vito; Michele, Lorenzo Di
      Journal of Physics: Condensed Matter (2019), 31 (7), 074003/1-074003/11CODEN: JCOMEL; ISSN:0953-8984. (IOP Publishing Ltd.)
      DNA nanostructures with programmable shape and interactions can be used as building blocks for the self-assembly of cryst. materials with prescribed nanoscale features, holding a vast technol. potential. Structural rigidity and bond directionality have been recognized as key design features for DNA motifs to sustain long-range order in 3D, but the practical challenges assocd. with prescribing building-block geometry with sufficient accuracy have limited the variety of available designs. We have recently introduced a novel platform for the one-pot prepn. of cryst. DNA frameworks supported by a combination of Watson-Crick base pairing and hydrophobic forces. Here we use small angle x-ray scattering and coarse-grained mol. simulations to demonstrate that, as opposed to available all-DNA approaches, amphiphilic motifs do not rely on structural rigidity to support long-range order. Instead, the flexibility of amphiphilic DNA building-blocks is a crucial feature for successful crystn.
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    34. 34
      Fabrini, G.; Minard, A.; Brady, R. A.; Di Antonio, M.; Di Michele, L. Cation-Responsive and Photocleavable Hydrogels from Noncanonical Amphiphilic DNA Nanostructures. Nano Lett. 2022, 22, 602– 611,  DOI: 10.1021/acs.nanolett.1c03314
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      34
      Cation-Responsive and Photocleavable Hydrogels from Noncanonical Amphiphilic DNA Nanostructures
      Fabrini, Giacomo; Minard, Aisling; Brady, Ryan A.; Di Antonio, Marco; Di Michele, Lorenzo
      Nano Letters (2022), 22 (2), 602-611CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Thanks to its biocompatibility, versatility, and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering, and drug delivery. Of particular importance for in vivo applications is the possibility of making such nanomaterials responsive to physiol. stimuli. Here, we demonstrate how combining noncanonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanostructures, which we termed "Quad-Stars", capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the hydrogel aggregates can be photo-disassembled upon near-UV irradn. in the presence of a porphyrin photosensitizer. The combined reversibility of assembly, responsiveness, and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.
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    35. 35
      Walczak, M.; Brady, R. A.; Mancini, L.; Contini, C.; Rubio-Sánchez, R.; Kaufhold, W. T.; Cicuta, P.; Di Michele, L. Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment. Nat. Commun. 2021, 12, 4743,  DOI: 10.1038/s41467-021-24989-7
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      35
      Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment
      Walczak, Michal; Brady, Ryan A.; Mancini, Leonardo; Contini, Claudia; Rubio-Sanchez, Roger; Kaufhold, William T.; Cicuta, Pietro; Di Michele, Lorenzo
      Nature Communications (2021), 12 (1), 4743CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Biol. has evolved a variety of agents capable of permeabilizing and disrupting lipid membranes, from amyloid aggregates, to antimicrobial peptides, to venom compds. While often assocd. with disease or toxicity, these agents are also central to many biosensing and therapeutic technologies. Here, we introduce a class of synthetic, DNA-based particles capable of disrupting lipid membranes. The particles have finely programmable size, and self-assemble from all-DNA and cholesterol-DNA nanostructures, the latter forming a membrane-adhesive core and the former a protective hydrophilic corona. We show that the corona can be selectively displaced with a mol. cue, exposing the 'Sicky' core. Unprotected particles adhere to synthetic lipid vesicles, which in turn enhances membrane permeability and leads to vesicle collapse. Furthermore, particle-particle coalescence leads to the formation of gel-like DNA aggregates that envelop surviving vesicles. This response is reminiscent of pathogen immobilization through immune cells secretion of DNA networks, as we demonstrate by trapping E. coli bacteria.
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    36. 36
      Zhang, D. Y.; Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 2009, 131, 17303– 17314,  DOI: 10.1021/ja906987s
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      36
      Control of DNA Strand Displacement Kinetics using Toehold Exchange
      Zhang, David Yu; Winfree, Erik
      Journal of the American Chemical Society (2009), 131 (47), 17303-17314CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA mols. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quant. predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.
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    37. 37
      Simmel, F. C.; Yurke, B.; Singh, H. R. Principles and Applications of Nucleic Acid Strand Displacement Reactions. Chem. Rev. 2019, 119, 6326– 6369,  DOI: 10.1021/acs.chemrev.8b00580
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      37
      Principles and Applications of Nucleic Acid Strand Displacement Reactions
      Simmel, Friedrich C.; Yurke, Bernard; Singh, Hari R.
      Chemical Reviews (Washington, DC, United States) (2019), 119 (10), 6326-6369CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Dynamic DNA nanotechnol., a subfield of DNA nanotechnol., is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addn., the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNA-based nanomachine (Yurke, B.; et al. Nature 2000, 406, 605-608). Since then, toehold-mediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnol. has grown exponentially. Besides mol. machines, the field has produced enzyme-free catalytic systems, all DNA chem. oscillators and the most complex mol. computers yet devised. Enzyme-free catalytic systems can function as chem. amplifiers and as such have received considerable attention for sensing and detection applications in chem. and medical diagnostics. Strand-displacement reactions have been combined with other enzymically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol.2015, 11, 287-294). Strand-displacement principles have also been applied in synthetic biol. to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnol. over the past years, the field now seems poised for practical application.
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    38. 38
      Stellwagen, E.; Lu, Y.; Stellwagen, N. C. Unified description of electrophoresis and diffusion for DNA and other polyions. Biochemistry 2003, 42, 11745– 50,  DOI: 10.1021/bi035203p
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      Unified Description of Electrophoresis and Diffusion for DNA and Other Polyions
      Stellwagen, Earle; Lu, Yongjun; Stellwagen, Nancy C.
      Biochemistry (2003), 42 (40), 11745-11750CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The electrophoretic mobilities and diffusion coeffs. of single- and double-stranded DNA mols. up to 50,000 bases or base pairs in size have been analyzed, using mobilities and diffusion coeffs. either measured by capillary electrophoresis or taken from the literature. The Einstein equation suggests that the electrophoretic mobilities (μ) and diffusion coeffs. (D) should be related by the expression μ/D = Q/kBT, where Q is the charge of the polyion (Q = zeo, where z is the no. of charged residues and eo is the fundamental electronic charge), kB is Boltzmann's const., and T is the abs. temp. If this equation were true, the ratio μ/zD should be a const. equal to eo/kBT (39.6 V-1) at 20°. However, the ratio μ/zD decreases with an increase in mol. wt. for both single- and double-stranded DNAs. The mobilities and diffusion coeffs. are better described by the modified Einstein equation μ/NmD = eo/kBT, where N is the no. of repeat units (bases or base pairs) in the DNA and m is a const. equal to the power law dependence of the diffusion coeffs. on mol. wt. The av. value of the ratio μ/NmD is 40±4 V-1 for 36 single- and double-stranded DNA mols. of different sizes, close to the theor. expected value. The generality of the modified Einstein equation is demonstrated by analyzing literature values for sodium polystyrenesulfonate (PSS). The av. value of the ratio μ/NmD is 35±6 V-1 for 14 PSS samples contg. up to 855 monomers.
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    39. 39
      Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Diffusion of macromolecules in agarose gels: Comparison of linear and globular configurations. Biophys. J. 1999, 77, 542– 552,  DOI: 10.1016/S0006-3495(99)76911-0
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      Diffusion of macromolecules in agarose gels: Comparison of linear and globular configurations
      Pluen, Alain; Netti, Paolo A.; Jain, Rakesh K.; Berk, David A.
      Biophysical Journal (1999), 77 (1), 542-552CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The diffusion coeffs. (D) of different types of macromols. (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in soln. and in 2% agarose gels to compare transport properties of these macromols. Diffusion measurements were conducted with concns. low enough to avoid macromol. interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromols. were analyzed sep. As chain length increases, diffusion coeffs. of DNA show a clear shift from a Rouse-like behavior (DG ≃ N0-0.5) to a reptational behavior (DG ≃ N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS soln. was estd. Diffusion coeffs. of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estn. of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromols. exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.
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      Schuck, P. Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport. Biophys. J. 1996, 70, 1230– 1249,  DOI: 10.1016/S0006-3495(96)79681-9
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      40
      Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport
      Schuck, Peter
      Biophysical Journal (1996), 70 (3), 1230-49CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The influence of mass transport on ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor, was investigated. A one-dimensional computer model for the mass transport of ligand between the bulk soln. and the polymer gel and within the gel was employed, and the influence of the diffusion coeff., the partition coeff., the thickness of the matrix, and the distribution of immobilized receptor were studied for a variety of conditions. Under conditions that may apply to many published exptl. studies, diffusion within the matrix was found to decrease the overall ligand transport significantly. For relatively slow reactions, small spatial gradients of free and bound ligand in the gel are found, whereas for relatively rapid reactions strong inhomogeneities of ligand within the gel occur before establishment of equil. Several types of deviations from ideal pseudo-first-order binding progress curves are described that resemble those of published exptl. data. Extremely transport limited reactions can in some cases be fitted with apparently ideal binding progress curves, although with apparent reaction rates that are much lower than the true reaction rates. Nevertheless, the ratio of the apparent rate consts. can be semiquant. consistent with the true equil. const. Apparently "cooperative" binding can result from high chem. on rates at high receptor satn. Dissocn. in the presence of transport limitation was found to be well described empirically by a single or a double exponential, with both apparent rate consts. considerably lower than the intrinsic chem. rate const. Transport limitations in the gel can introduce many generally unknown factors into the binding progress curve. The simulations suggest that unexpected deviations from ideal binding progress curves may be due to highly transport influenced binding kinetics. The use of a thinner polymer matrix could significantly increase the range of detectable rate consts.
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      Crank, J. the Mathematics of Diffusion, 2nd ed.; Oxford University Press: London, UK, 1979.
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      Semenov, S. N.; Markvoort, A. J.; Gevers, W. B. L.; Piruska, A.; de Greef, T. F. A.; Huck, W. T. S. Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions. Biophys. J. 2013, 105, 1057– 1066,  DOI: 10.1016/j.bpj.2013.07.002
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      Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions
      Semenov, Sergey N.; Markvoort, Albert J.; Gevers, Wouter B. L.; Piruska, Aigars; de Greef, Tom F. A.; Huck, Wilhelm T. S.
      Biophysical Journal (2013), 105 (4), 1057-1066CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Delineating design principles of biol. systems by reconstitution of purified components offers a platform to gauge the influence of crit. physicochem. parameters on minimal biol. systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films contg. immobilized substrates and reversible inhibitors. Quant. fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addn. of strong inhibitors, consistent with a math. model of mol. titrn. The results confirm that ultrasensitive and threshold effects at the mol. level can convert a graded input signal to a steep spatial response at macroscopic length scales.
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    43. 43
      Press, W. H.; Teukolsky, S. A.; Vetterling, W. T.; Flannery, B. P. Numerical Recipes in C: The Art of Scientific Computing, 2nd ed.; Cambridge University Press: Cambridge, MA, 1992; pp 656– 706.
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      Raue, A.; Kreutz, C.; Maiwald, T.; Bachmann, J.; Schilling, M.; Klingmüller, U.; Timmer, J. Structural and practical identifiability analysis of partially observed dynamical models by exploiting the profile likelihood. Bioinformatics 2009, 25, 1923– 1929,  DOI: 10.1093/bioinformatics/btp358
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      Structural and practical identifiability analysis of partially observed dynamical models by exploiting the profile likelihood
      Raue, A.; Kreutz, C.; Maiwald, T.; Bachmann, J.; Schilling, M.; Klingmueller, U.; Timmer, J.
      Bioinformatics (2009), 25 (15), 1923-1929CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
      Math. description of biol. reaction networks by differential equations leads to large models whose parameters are calibrated to optimally explain exptl. data. Often only parts of the model can be obsd. directly. Given a model that sufficiently describes the measured data, it is important to infer how well model parameters are detd. by the amt. and quality of exptl. data. This knowledge is essential for further investigation of model predictions. For this reason a major topic in modeling is identifiability anal. The authors suggest an approach that exploits the profile likelihood. It enables to detect structural non-identifiabilities, which manifest in functionally related model parameters. Furthermore, practical non-identifiabilities are detected, that might arise due to limited amt. and quality of exptl. data. Last but not least confidence intervals can be derived. The results are easy to interpret and can be used for exptl. planning and for model redn. Availability: An implementation is freely available for MATLAB and the PottersWheel modeling toolbox at http://web.me.com/andreas.raue/profile/software.html. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.
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      Kar, S.; Ellington, A. D. Construction of synthetic T7 RNA polymerase expression systems. Methods 2018, 143, 110– 120,  DOI: 10.1016/j.ymeth.2018.02.022
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      Construction of synthetic T7 RNA polymerase expression systems
      Kar, Shaunak; Ellington, Andrew D.
      Methods (Amsterdam, Netherlands) (2018), 143 (), 110-120CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
      T7 RNA polymerase (T7 RNAP) is one of the preferred workhorses for recombinant gene expression, owing in part to its high transcriptional activity and the fact that it has a small (17 base-pair), easily manipulated promoter. However, the high activity of the enzyme also often leads to an increased fitness burden on the host, limiting the predictability of its interactions and impact on host physiol., and potentially leading to mutations in constructs. Here we use a synthetic biol. approach to design and characterize a panel of T7 RNAP expression circuits with different modes of regulation that enable the reliable expression of downstream targets under a variety of conditions. First, we describe the construction of a minimal T7 RNAP expression system that is inducible by a small mol. anhydrotetracycline (aTc), and then characterize a self-limiting T7 RNAP expression circuit that provides better control over the amt. of T7 RNAP produced upon induction. Finally, we characterize a so-called T7 RNAP homeostasis circuit that leads to constitutive, continuous, and sub-toxic levels of T7 RNAP. Coupled with previously characterized mutant T7 RNAP promoters in vitro, we demonstrate that this modular framework can be used to achieve precise and predictable levels of output (sfGFP) in vivo. This new framework should now allow modeling and construction of T7 RNAP expression constructs that expand the utility of this enzyme for driving a variety of synthetic circuits and constructs.
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    46. 46
      Filonov, G. S.; Moon, J. D.; Svensen, N.; Jaffrey, S. R. Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution. J. Am. Chem. Soc. 2014, 136, 16299– 16308,  DOI: 10.1021/ja508478x
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      46
      Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution
      Filonov, Grigory S.; Moon, Jared D.; Svensen, Nina; Jaffrey, Samie R.
      Journal of the American Chemical Society (2014), 136 (46), 16299-16308CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small mols. in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although addnl. highly fluorescent RNA-fluorophore complexes would extend the utility of this technol., the identification of novel RNA-fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Addnl., aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here the authors describe a platform for rapid generation of highly fluorescent RNA-fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, the authors identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concns. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.
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    47. 47
      Dana, H.; Chalbatani, G. M.; Mahmoodzadeh, H.; Karimloo, R.; Rezaiean, O.; Moradzadeh, A.; Mehmandoost, N.; Moazzen, F.; Mazraeh, A.; Marmari, V.; Ebrahimi, M.; Rashno, M. M.; Abadi, S. J.; Gharagouzlo, E. Molecular Mechanisms and Biological Functions of siRNA. Int. J. Biomed. Sci. 2017, 13 (2), 48– 57
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      Molecular Mechanisms and Biological Functions of siRNA
      Dana Hassan; Rezaiean Omid; Moradzadeh Amirreza; Mazraeh Ali; Marmari Vahid; Rashno Mohammad Menati; Chalbatani Ghanbar Mahmoodi; Mahmoodzadeh Habibollah; Gharagouzlo Elahe; Karimloo Rezvan; Mehmandoost Narges; Moazzen Fateme; Ebrahimi Mohammad; Abadi Saeid Jan
      International journal of biomedical science : IJBS (2017), 13 (2), 48-57 ISSN:1550-9702.
      One of the most important advances in biology has been the discovery that siRNA (small interfering RNA) is able to regulate the expression of genes, by a phenomenon known as RNAi (RNA interference). The discovery of RNAi, first in plants and Caenorhabditis elegans and later in mammalian cells, led to the emergence of a transformative view in biomedical research. siRNA has gained attention as a potential therapeutic reagent due to its ability to inhibit specific genes in many genetic diseases. siRNAs can be used as tools to study single gene function both in vivo and in-vitro and are an attractive new class of therapeutics, especially against undruggable targets for the treatment of cancer and other diseases. The siRNA delivery systems are categorized as non-viral and viral delivery systems. The non-viral delivery system includes polymers; Lipids; peptides etc. are the widely studied delivery systems for siRNA. Effective pharmacological use of siRNA requires 'carriers' that can deliver the siRNA to its intended site of action. The carriers assemble the siRNA into supramolecular complexes that display functional properties during the delivery process.
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      Moreno-Risueno, M. A.; Benfey, P. N. Time-based patterning in development: The role of oscillating gene expression. Transcription 2011, 2, 124– 129,  DOI: 10.4161/trns.2.3.15637
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      Time-based patterning in development: The role of oscillating gene expression
      Moreno-Risueno Miguel A; Benfey Philip N
      Transcription (2011), 2 (3), 124-129 ISSN:.
      Oscillating gene expression is a mechanism of patterning during development in both plants and animals. In vertebrates, oscillating gene expression establishes the musculoskeletal precursors (somites), while in plant roots it establishes the position of future organs (lateral roots). Both mechanisms constitute a specialized type of biological clock that converts temporal information into precise spatial patterns. Similarities, differences, and their functionality in organisms that evolved independently are discussed.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2sbovVersw%253D%253D&md5=ca9baba56084bdb9d84951c6f1bbf4b7

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c06140.

    • All experimental, image analysis, numerical modeling and fitting methods; bright-field microscopy images; SAXS characterizations; I(r, t) patterning maps; data demonstrating control over domain thickness; analysis of the fitting model behavior; correlation between fitting parameters and their identifiability; performance of the extended Broccoli aptamer; patterning protocol for the RNA-synthesizing ACs; RNA synthesis in ACs; control experiments; nuclotide sequences; and supplementary video descriptions (PDF)

    • Large view of one patterning strand (p8) (AVI)

    • Zoomed-in view of one patterning strand (p8) (AVI)

    • Large view of two patterning strands (p1 and p8)(AVI)

    • Zoomed-in view of two patterning strands (p1 and p8)(AVI)

    • Large view of three patterning strands (p1, p6, and p8) (AVI)

    • Zoomed-in view of three patterning strands (p1, p6, and p8) (AVI)

    • Large view of five patterning strands (p1, p3, p5, p7 and p8) (AVI)

    • Zoomed-in view of five patterning strands (p1, p3, p5, p7 and p8)(AVI)

    • Large view of three patterning strands (p1, p6, and p8) and the stop strand (AVI)

    • Zoomed-in view of three patterning strands (p1, p6, and p8) and the stop strand (AVI)

    • Large view of one patterning strand (p1) (AVI)

    • Zoomed-in view of one patterning strand (p1) (AVI)

    • Broccoli RNA production and storage in artificial cells (example 1) (AVI)

    • Broccoli RNA production and storage in artificial cells (example 2) (AVI)

    • Zoomed-in view of Broccoli RNA production and storage in artificial cells (fluorescence) (AVI)

    • Zoomed-in view of Broccoli RNA production and storage in artificial cells (bright-field and light fluorescence) (AVI)


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