Overlay of HPLC traces of setups 2–4.

<p>(<b>A</b>) Setup <b>2</b>, 2-MeImpU, -dG; Setup <b>2a</b> -2-MeImpU, -A, -dG. (<b>B</b>) Setup <b>3</b>, 2-MeImpU, -A, -dG. (<b>C</b>) Setup 5, 2-MeImpU, -C, -dG. Grey boxes indicate FP+3 product fractions analyzed by MALDI-TOF MS.</p

Product accumulation after dG incorporation.

<p>HPLC chromatograms of setup <b>2</b> reaction mixtures after 14 days incubation at -18.4°C: black stipples, *pU, *pG; cyan, *pU, *pG/*pdG (1:1); blue, *pU, *pdG. Red: ligation of ImpUUG as internal control for the elution range of FP+3 products (using 2.5 µM <b>FP</b> and 7.5 µM ImpUUG, see Supporting Materials and Methods in File S1 for synthesis of ImpUUG). Arrows indicate accumulated products due to dG insertions; red: non-specific insertion, green: template-directed ones.</p

Analysis of FP+3 products in setup 2 and 2a.

<p>Oligomers identified by MALDI-TOF MS (<b>A</b>) Setup <b>2</b> – (I): FP-UUÛ (3344.26 g/mol, found m/z 3343.46, trace); FP-UUdG (3367.26 g/mol, found m/z 3365.86, and Na⁺-adducts) (II). FP-UUdG (3367.26 g/mol, found m/z 3367.63). (<b>B</b>) Setup <b>2a</b> – (I): FP-UUÛ (3344.26 g/mol, found m/z = 3343.46, trace); FP-UUdG (3367.26 g/mol, found m/z 3365.86), FP-UÂdG (3390.33 g/mol, found m/z 3389.08) (II). FP-UUdG (3367.26 g/mol, found m/z 3367.63); FP-UÂdG (3390.33 g/mol, found m/z 3390.66).</p

MALDI-TOF MS of FP+3 products in setup 3.

<p>Identified oligomers: FP-UAdG (3390.33 g/mol, found m/z: 3390.77 (I) and 3390.56 (II), FP-UUdG (3367.26 g/mol, found m/z: 3368.30 (I) and 3367.40 (II). FP-UUU (3344.26 g/mol, found m/z: 3343.46, trace in (I)).</p

Experiment overview.

<p>6-FAM fluorescently labeled primers (FP) bound to template <b>t</b>_<b>n</b> were incubated with mixtures of activated monomers (*pNs) at -18.4 °C. As emphasized in the schematic, the copying a motif of three template residues (blue) was investigated. The <b>t</b>_<b>1</b> motif is highly conducive to enzyme-free primer elongation, while the underlined motifs in <b>t</b>_<b>2</b>-<b>t</b>_<b>4</b> are the sequences reported to block such reactions. Green spot = 6-FAM marker, small letters = deoxynucleotides (only in sequence drawings). B = nucleobase.</p

Permeability coefficients calculated from the release of entrapped CF at 45°C.

<p>Permeability coefficients calculated from the release of entrapped CF at 45°C.</p

Preparation of fatty acid vesicles encapsulating CF, visualized by epifluorescence microscopy.

<p>(A) Micrograph showing the formation of vesicular structures of polydisperse sizes and diverse degree of lamellarity from an aqueous solution of 21 mM LA in 100 mM bicine buffer, pH 7.95. (B) Another LA vesicle suspension, but 1 h after extrusion through polycarbonate filters of 400 nm pore diameter. (C) LA vesicles of monodisperse size just after size exclusion chromatography. Once the non-entrapped material has been removed, vesicles retaining CF exhibit a characteristic green color. (D) The fluorescence spreads throughout the sample after addition of a detergent such as Triton-X100. Samples A and B are stained with the lipophilic dye Nile red. All panels are at the same magnification.</p

3D bifurcation diagram.

<p>Combined bifurcation diagram, showing the dependence of the stationary solutions on both the degradation rate constants (, and ), and the rate constants for uptake of nutrients (, and ). Three different regions are distinguished in the parameter space: (i) For , only a residual steady state (red surface) is possible regardless of the values of , and . (ii) In the range the number of possible attainable steady states is, in contrast, dependent of the values of , and . The star shows the location of the critical point obtained for , and when . (iii) For , there is again only one possible steady state, regardless of the values of , and . However, is still strongly dependent on the values of these incorporation rate constants, which remain critical to relate this steady state with a proper functional steady state or, instead, with some residual functioning of the system.</p

Experimental release profiles of CF from vesicles at 45°C.

<p>(A) Time evolution of the concentration of released CF, normalized to the concentration at equilibrium, i.e. after final addition of detergent. The resulting exponential curves were linearized in a semi-logarithmic plot for the first 30 min (B). Linear tendencies are obtained in agreement to Eq. 4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039480#pone.0039480.s002" target="_blank">Text S2</a>, identifying the slope as the rate constant of release . (); (). Red triangles: vesicles made of LA. Blue circles: vesicles made of OA/GMO in a molar ratio 2∶1.</p

Values of rate constants and fixed concentrations in the kinetic protometabolic model.

<p>All parameters have the same values as already defined in previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039480#pone.0039480-Piedrafia1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039480#pone.0039480-Piedrafita1" target="_blank">[39]</a>, except those marked with an asterisk, which are new.</p