Puregene protein precipitation solution

O-(Pyridin-3-yl-methyl)hydroxylamine (PMOA), butyraldehyde, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (Tris), EDTA, DNase I (type IV, bovine pancreas), alkaline phosphatase (Escherichia coli), nuclease P1 (Penicillium citrinum), RNase A (bovine pancreas), RNase T1 (Aspergillus oryzae), and proteinase K (Tritirachium album) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphodiesterase I (Crotalus adamanteus venom) and adenosine deaminase were purchased from Worthington Biochemical Corp. (Lakewood, NJ). LC-MS grade solvents were purchased from Fisher Chemical Co. (Pittsburgh, PA). Puregene protein precipitation (PP) solution and DNeasy Blood & Tissue kit were purchased from Qiagen (Germantown, MD). N,N-Bis(2-chloroethyl)ethylamine hydrochloride was purchased from Aaron Chemistry Gmbh (Mittenwald, Germany). Pall 10 kDa filter with omega membrane, MWCO 10 kDa, was purchased from Sigma Aldrich (St. Louis, MO) All other chemicals were ACS grade and purchased from Sigma-Aldrich unless otherwise stated. Strata-X 33 μm Polymeric Reversed Phase 30 mg solid-phase extraction (SPE) cartridge was purchased from Phenomenex (Torrance, CA). Sola SCX cartridges were purchased from Thermo Scientific (Bellafonte, PA). MicroLiter autosampler vials with silanized glass inserts were purchased from Wheaton (Millville, NJ).

The experiments conducted with a single-stranded oligonucleotide are reported in supporting information. AP sites were prepared by uracil DNA glycosylase-catalyzed removal of uracil in double-stranded (5′-GCCGTUAGGTA-3’ • 3’-CGGCAATCCAT-5′) oligonucleotide.^{22 (link)} The oligonucleotide was reacted with 5 mM PMOA at 37 °C at pH 7.0 without a catalyst, or at pH 7.4 with 5 or 10 mM L-histidine.
To explore the catalytic effect of enzymes, the double-stranded 11-mer oligonucleotide (50 μL, 1.89 μg/μL), RNase A (15 μL, 150 μg), and RNase T₁ (1 μL, Sigma R1003) were added to 500 μL of TE buffer [50 mM Tris-HCl buffer, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0]. PMOA (25 μL, 100 mM in water) was added and incubated for 30 min at 37 °C. Then, proteinase K (20 μL, 400 μg) was added and incubated for another 30 min. Finally, Puregene Protein Precipitation (PP) solution (250 μL, pH = 8, Qiagen) was added, and the mixture was incubated for an additional 30 min. At selected time points, aliquots were taken, and the unreacted oligonucleotide was quantitated with HPLC-UV at 260 nm.