Cuso4 5h2o

All bacterial strains used in this work were isolated from a consortium associated with the Antarctic ciliate E. focardii and identified as Marinomonas, Rhodococcus, Pseudomonas, Brevundimonas, and Bacillus [17 (link),18 (link),20 (link)]. All strains were grown at 22 °C on agarized or liquid Luria–Bertani (LB) medium (tryptone 10g/L, yeast extract 5 g/L, and NaCl 5 g/L). All media were purchased from Liofilchem. Analytical grade copper (II) sulfate pentahydrate salt, CuSO₄·5H₂O, and the other chemicals were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).

NaNO₃ (PanReac, Barcelona, Spain) and NaH₂PO₄·2H₂O (Honeywell, Tokyo, Japan) were used as nitrogen and phosphorus sources, respectively, while the trace elements Na₂EDTA (Sigma, St. Luis, MO, USA), FeCl₃·6H₂O (Acros Organics, Geel, Belgium), CuSO₄·5H₂O (Sigma), ZnSO₄·7H₂O (Sigma), CoCl₂·6H₂O (Fisher Scientific, Waltham, MA, USA), MnCl₂·4H₂O (Acros Organics), and Na₂MoO₄·2H₂O (Chem-Lab NV, Zedelgem, Belgium) were used for media preparation. Cyanocobalamin, Thiamine HCl, and Biotin were procured from Sigma-Aldrich. Chemicals used for analysis included ammonium bicarbonate (Sigma), HPLC-grade chloroform (Honeywell) and HPLC-grade methanol (Fisher Scientific).

Reagents Trizma base; NAD⁺; xanthine; CuSO₄∗5  H₂O were purchased from Sigma Aldrich (Poznan, Poland). Determinations were performed using a UV/VIS Lambda 40P (Perkin Elmer). Extinction changes were recorded for 5 minutes at 30°C. The enzymatic activity was measured as formation of uric acid and NADH (increases in A₃₄₀ and A₃₀₂) and expressed in mU/mL (milliunits per milliliter). The enzymatic activity was calculated taking into account the initial rates of reaction. Uric acid formation was measured at 302 nm (isoforms XDO and XO) because its absorbance is still high there, whereas changes in NAD⁺ concentration do not contribute. During the calculation of isoforms activity of xanthine oxidoreductase included molar extinction coefficients: NADH + H⁺  
ε340 = 6.22 × 103 L mol⁻¹ × cm⁻¹; NADH + H⁺  
ε302 = 2.30 × 10³ × mol⁻¹ cm⁻¹ [8 (link), 9 ].

A 1.0 × 10⁻²mol L⁻¹ stock solution of luminol (3-aminophthalhydrazide) was prepared by dissolving luminol (Sigma) in a 0.1 mol L⁻¹ sodium hydroxide solution and stored at 4 °C. Working solutions of luminol were prepared by diluting the stock solution with ultra-pure water. Bovine serum albumin (BSA) was purchased from Sigma Sangon Biotech Co., Ltd. (Shanghai, China). CuSO₄·5H₂O, sodium hydroxide, isopropanol, Triton X-100, cholesterol and ChOx were purchased from Sigma-Aldrich Co., Ltd. (USA). A stock solution of cholesterol was prepared by dissolving cholesterol in a mixture of isopropanol and Triton X-100 (1:1, v/v), and the standard cholesterol solutions were diluted with PBS (pH 7.4) and then stored at 4 °C. All of the reagents were used as purchased without purification, and ultra-pure water was used throughout.

Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and CuSO₄.5H₂O were bought from Sigma-Aldrich. Stock solutions of PDC-click precursors (PDC-4,2-Alk, PDC-4,3-Alk, PDC-4,0-N3 and PDC-4,PEG-N3) and 5-BrdU-click partners (5-BrdU-Alk and 5-BrdU-N3) were prepared in DMSO at 1 mM concentration, and stored in the dark at −20°C. For all the following experiments, copper-catalyst (CuSO₄.5H₂O), THPTA and sodium ascorbate solutions were freshly prepared in ultrapure H₂O at respectively 10 mM for the CuSO₄.5H₂O solution, 50 mM for the THPTA solution and 40 mM for the sodium ascorbate solution. Oligonucleotide sequences were purchased from Eurogentec as dried samples purified by HPLC-RP. Oligonucleotides were dissolved in MilliQ water at 200 μM concentration and stored at −20°C. Oligonucleotide sequences used for click reaction in situ are the following: c-Myc22 (G14T-G23T): TGAGGGTGGGTAGGGTGGGTAA, 22AG: AGGGTTAGGGTTAGGGTTAGGG and TERRA: AGGGUUAGGGUUAGGGUUAGGG.
Oligonucleotides were folded and CuAAC reactions were carried out in K10 buffer (10 mM Li.Caco pH 7.2, 10 mM KCl, 90 mM LiCl).
Solid-phase extraction (SPE) was performed with Clarity® OTX (Phenomenex) columns. To facilitate elution of the fractions, a manifold device as vacuum source was employed (P = 0.4 Bar).

To mitigate the effects of rolling lines and impurities associated with commercial foils, high-purity electroplated copper substrates were fabricated in-house. A thin layer (100 nm) of copper strike layer was sputtered (Lesker, 99.999%) onto RCA-cleaned SiO₂ wafers in a cleanroom facility. 30 µm of copper was subsequently electroplated onto the strike layer in an acidic electroplating bath consisting of 94 g/L CuSO₄∙5H₂O (Sigma Aldrich, ≥98.0%) and 100 ml/L H₂SO₄ (Merck, 95–97%). Copper electroplating in a sulfuric acid bath is highly selective towards copper deposition, but nevertheless, low current density DC plating was employed to avoid co-deposition of potential impurities from the solution. The plated films were thoroughly rinsed in deionized water and dried with a combination of acetone and isopropanol rinsing and blow-drying under N₂ flow. Finally, the foils were delaminated from the SiO₂ wafer immediately before inserting them into the growth chamber, revealing an atomically flat surface (Supplementary Fig. 5), onto which our growths were subsequently carried out^{17 (link), 49 (link), 50 (link)}.