Amine peg2 biotin

Poly(ethylene terephthalate) (PET) surgical meshes (PETNF203) were purchased from Textile Development Associates, Inc. (Brookfield, CT, USA). Avidin-coated microspheres (CP01N) were purchased from Bangs Laboratories, Inc. (Fishers, IN, USA), sulfosuccinimidyl-4-O-(4,4-dimethoxytrityl) butyrate (Sulfo-SDTB) was obtained from bioWORLD (Dublin, OH, USA). Biotin N-hydroxysuccinimide ester was purchased from AppliChem (Darmstadt, Germany). Amine-terminated poly(ethylene glycol) dimer functionalized with biotin (Amine-PEG₂-Biotin) and the Alexa Fluor 647 Microscale Protein labeling kit were purchased from Thermo Fisher Scientific (Dreieich, Germany). All other chemicals were purchased from Sigma-Aldrich (Darmstadt, Germany). Chemical reagents and solvents used for the synthesis were of analytical grade and used as received. All synthesis experiments were carried out in air. Water was deionized with a Milli-Q Reference water purification system manufactured by Millipore Corporation (Darmstadt, Germany). All reactions involving the modification of PET were carried out in a Snycore^®® Reactor R-24 (Büchi, Flawil, Switzerland). Scanning electron microscopy (SEM) was performed on a Quanta 250 FEG microscope using an accelerating voltage of 20 kV in high vacuum from FEI (Hillsboro, OR, USA). The micrographs were then analyzed with xT Microscope Control (4.1.4).

DNA fragments harboring 8-oxoG were enriched as previously described^{37 (link)}, with minor modifications. Briefly, 5 µg of the genomic DNA extracted from adipose or lung tissues of randomly selected mice (n = 2) were sheared with an S2 ultrasonicator (Covaris, Woburn, MA, USA) in 10 mM Tris buffer (pH 8.0) to obtain ~150-bp fragments. After sonication, the fragmented DNA was concentrated to 20 µl in 100 mM NaPi buffer (pH 8.0) using QIAquick PCR purification kit (Qiagen). A 100-µl volume of 100 mM NaPi buffer containing 20 mM amine-PEG2-biotin (Thermo Fisher Scientific) was added and the mixture was heated to 75 °C for 10 min. After thermal equilibration, 5 mM K₂IrBr₆, a mild one electron oxidant, was added for 1 h for 8-oxoG biotinylation through covalent adduction. The DNA fragments biotinylated at 8-oxoG were eluted with 125 µl Tris buffer using the QIAquick PCR purification kit and extracted using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific); strands complementary to those with bound biotinylated 8-oxoG were released by incubation in 150 mM NaOH at 20 °C for 30 min, and concentrated to 10 µl ddH₂O using ssDNA/RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA).

DNA fragments with 8-oxoG were purified as previously described [41 (link)]. Briefly, 5 µg of the genomic DNA extracted from sperm was fragmented with an S2 ultrasonicator (Covaris, Woburn, MA, USA) in 10 mM Tris buffer (pH 8.0) to obtain approximately 150-bp fragments. After sonication, the fragmented DNA was concentrated to 20 µL in 100 mM NaPi buffer (pH 8.0), using QIAquick PCR purification kit (Qiagen). A 100 µL volume of 100 mM NaPi buffer with 20 mM amine-PEG2-biotin (Thermo Fisher Scientific) was added and the mixture was heated to 75 °C for 10 min. After thermal equilibration to room temperature, 5 mM K₂IrBr₆ (Sigma-Aldrich) was added for 8-oxoG biotinylation. The biotinylated DNA fragments were eluted with 125 µL Tris buffer using the QIAquick PCR purification kit and collected using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). Complementary DNA strands to those with bound biotinylated 8-oxoG were released by incubation in 150 mM NaOH at 20 °C for 30 min and concentrated to 10 µL ddH₂O using the ssDNA/RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA).