Piperidine

Fmoc-protected amino acids and Wang resin were purchased from Novabiochem®. OymaPure®, N-ethyldiisopropylamine, piperidine and trifluoroacetic acid were obtained from Carl Roth. Dimethylformamide (DMF for peptide synthesis), diethyl ether and dimethylsulfoxid was purchased from Acros Organics. Acetonitrile was purchased from Fisher Scientific. Uranyl acetate and fluorescamine were purchased from Merck. Lysozyme was purchased from Amresco. PBS, Thioflavin T and α-cyano-4-hydroxycinnamic acid were purchased from Sigma Aldrich. Proteostat® was purchased from Enzo Life Sciences. All chemicals are listed in Supplementary Data 1 and were used as received unless explicitly stated otherwise.

Growth conditions for H. polymorpha were indicated above. In vivo footprinting method for Aspergillus nidulans [47 (link)] was adapted to H. polymorpha. After extraction of methylated DNA, a 10-fold diluted piperidine (Roth, Germany) was added to each DNA aliquot. Samples were incubated for 3 minutes at 90°C and cooled on ice. In vitro methylation was performed by dissolving DNA in 200 μl of MES buffer and 1 μl of DMS and incubating the mixture at 90°C for 90 s. After ethanol precipitations, DNA was resuspended in TE at final concentration of 0.7 mg/ml. A sample DNA aliquot was loaded on agarose gel to confirm the existence of a smear containing fragments in a range of 0–1000 bp. Methylated and piperidine treated DNA samples were analysed by LMPCR [47 (link)], using Vent polymerase (NEB). [³²P]ATP-labelled amplification products were separated on a sequencing gel which was dried and exposed to intensifying screens. To investigate the H. polymorpha YNT1 promoter, Primer1_sense, Primer2_sense, Primer3_sense were used (S1 Table).

Most of the used reagents (1,4-diaminobutane, N-(3-fimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), citric acid (CA), 1,4-butanediol diglycidyl ether (BDDE), α-cyano-4-hydroxycinnamic acid (HCCA), and Fmoc-protected amino acids) were purchased from Sigma-Aldrich Poznan, Poland. Hyaluronic acid (HA) was obtained from Contipro a.s., Dolni Dobruc, Czech Republic (product name: HyActive; molecular weight: 80–130 kDa). NaOH and HCl were obtained from Eurochem BGD Sp. z o.o., Tarnow, Poland. N,N-Diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), and piperidine were purchased from Carl Roth GmbH + Co. KG, Karlsruhe, Switzerland. Trifluoracetic acid (TFA), needed for the preparation of TA30 (0.1% TFA), was obtained from Fluorochem Ltd., Hadfield, United Kingdom. The reagents used during peptide synthesis, Fmoc-protected amino acids and 2-chlorotrityl resin, were purchased from Sigma-Aldrich Poznan, Poland. Dimethylformamide (DMF) was obtained from Avantor Performance Materials Poland S.A., Gliwice, Poland. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium-4-toluene sulfonate (DMT/NMM/TosO-) was developed at and obtained from the Institute of Organic Chemistry, Technical University of Lodz, Poland.
All prepared samples of the carbon nonwovens had dimensions of 2 × 2 cm.