Abstract. Neural networks have been trained to predict the subcellular location of proteins in prokaryotic or eukaryotic cells from their amino acid composition.
SUPPLEMENTARY FIGURES
Figure S1 1
Figure S1. CD spectra of selected PQSs folded in the absence or presence of stabilizing cation. CD spectra of oligonucleotides representing human (blue/black lines) and zebrafish (red/green lines) PQSs present in PPRs of the seven selected genes (apba1, col2a1, fzd5, map3k1, nog3, nr2e1 and shox2) and two control genes negative for ThT assay (jun and gnaq) folded in the absence (red/blue lines) or in the presence (green/black lines) of 100 mM KCl.
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Figure S2. CD spectra of wild type and mutated PQSs for col2a1, fzd5 and nog3. CD spectra of oligonucleotides representing wild type (green/black lines) and mutated (red/blue lines) human (blue/black lines) and zebrafish (red/green lines) PQSs present in PPRs of col2a1, fzd5, nog3 genes.
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Figure S3. CD spectra of col2a1, fzd5 and nog3 PQSs in the presence of TMPyP2 or TMPyP4 drugs. CD spectra of oligonucleotides representing PQSs present in PPRs of zebrafish col2a1, fzd5, nog3 in the presence of 30 μM TMPyP2 (red lines) or in the presence of 30 μM TMPyP4 (black lines).
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Figure S4. Assays to test the effect of ASOs on G-quadruplexes - duplex DNA equilibrium and triplex formation. A. Sequences of oligonucleotides representing PQSs present in PPRs of zebrafish col2a1, fzd5, nog3 and their corresponding ASOs. B. PAGE of 5’-32Plabelled oligonucleotides representing PQSs folded as G-quadruplexes in the presence of the corresponding ASOs or pre-folded as G-quadruplexes and then incubated with the
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corresponding ASOs for 1 or 20 h at 28°C. ASOs were used in PQS:ASO molar relationships of 1:1, 1:10 and 1:100. 5’-32P-labelled oligonucleotides representing PQSs folded as Gquadruplexes were used as mobility controls and CTRL was used instead of ASO as specificity control at the higher molar relationship (1:100). C. Bar graph of fluorescence enhancement (F/F0) of ThT in the presence of oligonucleotides representing PQSs folded as G-quadruplexes in the presence of the corresponding ASOs in PQS:ASO molar relationship of 1:1. Oligonucleotides representing PQSs folded as G-quadruplexes were used as controls and CTRL was used instead of ASO as specificity control at the same molar relationship (1:1). D. PAGE of duplex DNA formed by annealing of 5’-32P-labelled oligonucleotides representing PQSs with the corresponding ASO, incubated with the corresponding ASO in duplex:ASO molar relationships of 1:1, 1:10 and 1:100 for 20 h at 28°C. 5’-32P-labelled single strand oligonucleotides representing PQSs were used as mobility controls.
Supplementary Methods for Figure S4: PAGE assays To test the effect of ASOs on G-quadruplexes - duplex DNA equilibrium, G-quadruplexes forming oligonucleotides were 5’-end-labelled by phosphorylation with [γ-32P] ATP using T4 polynucleotide kinase (Fermentas) following manufacturer procedures. Labelled oligonucleotides were purified by gel-filtration chromatography using Micro Bio-Spin™ 6 Columns (Bio-Rad). Labelled oligonucleotides (≈ 0.05 μM) were folded in 10 mM Tris-HCl, pH 7.5, 100 mM KCl by heating at 95°C for 5 min and slowly cooling to room temperature over 2 h, in the presence (for assays during folding) or the absence (for assays posterior to folding) of the corresponding ASO or CTRL in 0.05, 0.5 or 5 μM. For assays posterior to 6
folding, the corresponding ASO or CTRL were added to the folded labelled oligonucleotides and incubated for 1 or 20 h at 28°C. To test the effect of ASOs on triplex formation, duplex DNA were formed by heating a mixture of labelled G-quadruplexes forming oligonucleotides with the corresponding ASO (≈ 0.05 μM each) at 95°C for 5 min and slowly cooling to room temperature during 2 h in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM EDTA. Then, the corresponding ASO or CTRL were added in 0.05, 0.5 or 5 μM and incubated for 20 h at 28°C. Reactions were resolved into 15 % nondenaturing
polyacrylamide
gel
electrophoresis
(PAGE)
in
0.5
X
TBE
buffer
[Tris/borate/EDTA (1 X TBE: 45 mM Tris-borate, pH 8.0, 1 mM EDTA)] at 10 mA and 4°C. Gels were heat-vaccum dried, exposed to X-Ray films (CL-X Posure TM Film, Thermo Scientific), developed and registered using HP Scanjet 3770 scanner.
ThT fluorescence assays For assaying the effect of anti-sense oligonucleotides (ASOs) on the G-quadruplexes duplex DNA equilibrium, ASOs or nonspecific control (CTRL) were incubated with Gquadruplex forming oligonucleotides in equimolar concentrations (2 μM each) during the folding step, and then were mixed with ThT to perform fluorescence measurements.
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Figure S5. Determination of the highest sub-toxic dose of ASO for embryo microinjection. CTRL oligonucleotide was injected in several dilutions in KCl 0.1 M ranging from 0.001 to 100 ng/µl in order to determine the highest sub-toxic dose that did not produced evident mortality, developmental malformations or delays compared to control embryos injected with KCl 0.1 M. Percentages of dead, deformed and normal embryos were determined at 24-hpf and 48-hpf to define 5 ng/µl as the highest sub-toxic dose of ASO.
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Figure S6. Temporal expression profile of col2a1, fzd5 and nog3 during zebrafish development. The relative abundance of nog3, col2a1 and fdz5 transcripts was measured by RT-qPCR in embryos at different developmental stages ranging from 1-2-cell to 6-dpf. Bars represent the mean of the three technical repeats relativized to the 1-2-cell stage and error bars correspond to standard deviation (SD).
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Figure S7. Analysis of cartilage pattern and myoD/krox20 expression pattern in col2a1ASO injected embryos. A. Alcian blue staining of craniofacial cartilage in 4-dpf staged larvae injected with CTRL (i) or col2a1-ASO (ii). Ventral views, anterior to the left. B. WISH assessing the expression of myoD mRNA as a marker of somites and krox20 (erg2) mRNA as a marker of rhombomeres 3 and 5 in 10-somite staged embryos injected with CTRL (i) or col2a1-ASO (ii). In lateral views anterior is to the left, and in dorsal views anterior is up. Numbers and percentages of embryos/larvae with phenotype are indicated in each panel. Scale bars (200 μm) are represented in Ai and Bi. cb 3-7: ceratobranchial cartilages 3 to 7; ch: ceratohyal cartilage; hs: hyosymplectic cartilage; m: Meckel’s cartilage; pf: pectoral fin; pq: palatoquadrate cartilage; r3/5: rhombomeres 3 and 5; s: somites.
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Figure S8. Analysis of tbx2b expression pattern in fzd5-ASO injected embryos. WISH assessing the expression of tbx2b mRNA as a marker of ventral diencephalon, eyes, trigeminal ganglion, and otic vesicles in 10-somite (A-B) and 15-somite (C-D) staged embryos injected with CTRL (A, C) or fzd5-ASO (B, D). In lateral views anterior is to the left, and in fronto-dorsal views dorsal is up. Numbers and percentages of embryos/larvae with phenotype are indicated in each panel. Scale bars (200 μm) are represented in A and C. e: eyes; ov: otic vesicles; tg: trigeminal ganglion; vdc: ventral diencephalon.
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Figure S9. Rescue of the zebrafish embryos phenotype produced by nog3-ASO injection by coinjection with nog3-mRNA. Representative pictures of Alcian Blue staining of craniofacial cartilages along with box-plots of the relative ceratohyal cartilages angle and ceratobranchial cartilages number of 4-dpf staged larvae injected with CTRL, CTRL + nog3mRNA, nog3-ASO and nog3-ASO + nog3-mRNA. * p
