Identification and design of CPP-like sequences.

<p>(A) Methodology used to identify and design CPP-like sequences. First, a random forest predictor is trained on a set of known CPP and non-CPP sequences. The resulting probabilistic model is then used to identify CPP-like sequences and converted into an energy measure (E = 1 –P_CPP(S)) for novel peptide design by simulated-annealing optimization (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004786#sec008" target="_blank">Materials and Methods</a>). (B) Algorithm performance during the design of CPP-like sequences. 32 random peptide sequences of length 16 were optimized with varying fragment lengths for 1000 iterations. Short fragments of more than 4 amino acids proved sufficient to transform non-CPP sequences to CPP-like sequences.</p

Minimal inhibitory concentrations for the tested peptides.

<p>Minimal inhibitory concentrations for the tested peptides.</p

Designed peptides internalized and reach nuclear localization in mouse cells.

<p>Primary cultured MEF cells were exposed for 20 min to 3 designed CPPs labeled with TAMRA at the N-terminus (TAMRA-α-NLS-C, TAMRA-NLS-α-CE and TAMRA chimera; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004786#pcbi.1004786.t001" target="_blank">Table 1</a>) or their respective control peptides (TAMRA-α-NLS-C woRK, TAMRA-NLS-α-CE woRK and TAMRA-chimera woRK; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004786#pcbi.1004786.t001" target="_blank">Table 1</a>). All peptides were used at a final concentration of 60 μM. To evaluate the co-localization of these peptides with nuclear DNA, cells were stained with DAPI after 20 min. The figure shows the fluorescence observed in DAPI, TAMRA and merged images to show the co-localization of DAPI and TAMRA signals; the squares indicate the area magnified to the right. The scale bar represents 100 μm.</p

DNA binding.

<p>(A) Quantification of DNA retention obtained from three independent electrophoretic mobility shift assays. All measurements are shown and horizontal bars denote the sample mean. Representative gels are shown in (B-C).</p

Pheromone activity.

<p>The pheromone signaling pathway is confirmed by the expression of Fus1-GFP, characteristic “shmoo” phenotype and the absence of budding observed in differential interference contrast (DIC) bright field. Concentrations were 20 μM for α-factor and 60 μM for all other peptides. The negative control was water without any peptide.</p

Designed peptides internalized into yeast cells.

<p>(A-F) Internalization assays in BY4741 yeast cells for peptide controls where Arg and Lys residues were replaced by Glu residues (A-C) and the original peptides (D-F). All peptides were used at 60 μM (final concentration) and labeled with the TAMRA fluorophore (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004786#pcbi.1004786.t001" target="_blank">Table 1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004786#sec008" target="_blank">Materials and Methods</a> for further details).</p

Peptides used in this study.

<p>Peptides used in this study.</p

Antibacterial activity.

<p>DH5α <i>E</i>. <i>coli</i> cells were treated with varying concentrations of peptides ranging from 0 to 16 μM for the peptides and 0 to 48 μM for Ampicillin. The relative area under the growth curve measured the growth inhibition. Measurements from four repetitions distributed onto two different plates are shown as dots colored by their respective concentrations. Linear regressions are shown as blue lines and their respective standard errors as blue shades. Dashed lines indicate the mean area under the curve for the H2O controls and its 50% value respectively. Minimal inhibitory concentrations to achieve 50% or 100% of cell growth inhibition are reported.</p

CD spectra of peptides.

<p>Spectra of peptides in water (blue line) and 50% TFE (red line) for NLS-α (A), NLS-α-CE (B), NLS-CE (C) and chimera (D) are presented.</p