S1 Supporting Information for Pyrroloquinoline quinone maintains redox activity when bound to a DNA aptamer Ismaila Emahi, Isabel M. Mulvihill, and Dana A. Baum* Department of Chemistry, Saint Louis University, St. Louis, Missouri 63103, USA * To whom correspondence should be addressed. D.A.B. – Tel: +1 314 977 2842; Fax: +1 314 977 2521; Email: dbaum1@slu.edu. Supplemental Materials and Methods Reagents and materials. DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT, Coralville, IA) and purified by denaturing polyacrylamide gel electrophoresis (PAGE) using 1X TBE running buffer (89 mM each of Tris and boric acid, 2 mM EDTA, pH 8.3). Samples were extracted from the gel by the crush-and-soak method in TEN buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 300 mM NaCl), recovered by ethanol precipitation, and quantified by UV absorbance. 1 The single-stranded DNA (ssDNA) random pool for in vitro selection was 5’-GAACTAGATCGCAGC-N 70 -GGATCGAGGTAATCC-3’. The N 70 designation indicates 70 nucleotides with equimolar incorporation of the four DNA bases A, C, G and T at each position. Two primers, primer 1: 5’-CAACAACAACAA-X- GGATTACCTCGATCC-3’ and primer 2: 5’–GAACTAGATCGCAGC-3’, anneal to the 3’ and 5’-ends of the random pool, respectively, during PCR amplification. The non-amplifiable spacer 18 linker in primer 1 (designated as X) allows for separation of the inactive complement from the active pool sequence via PAGE following PCR. The sequence of the unstructured DNA oligo used in the control experiments was 5’-GAACTAGATCGCAGC-(CAA) 23 - CGGATCGAGGTAATCC-3’. PQQ was purchased from Berry & Associates, Inc (Dexter, MI). 2,6-dichlorophenolindophenol (DCPIP) was purchased from Fisher Science Education (Nazareth, PA). Sodium ascorbate was obtained from Sigma-Aldrich Co. (St. Louis, MO). In vitro selection. DNA aptamers for PQQ were identified through in vitro selection (Figure S1) using the random pool described above. PQQ was coupled to MagnaBind amine-derivatized beads (Thermo Fisher) using 50 mM 4-(4, 6-Dimethoxy-1, 3, 5-triazin-2-yl)-4- methylmorpholinium (DMT-MM) in 100 mM MOPS pH 7.0 and 1 M NaCl. The amount of PQQ coupled to the beads was determined spectroscopically by measuring the A 330 of the coupling solution before and after the coupling reaction. Prior to each incubation with beads, the DNA pool was annealed by heating to 95 °C for 3 minutes, followed by incubation on ice for 5 min in 1X HEPES annealing buffer (5 mM HEPES pH 7.5, 10 mM NaCl, and 0.1 mM EDTA). Selections were initiated by first incubating 200 pmol of the random pool with amine-derivatized beads that had not been coupled to PQQ. This pre-selection column was intended to remove sequences that bind to the beads or the tether. The unbound sequences in the supernatant were Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2014