Synthesis and Characterization of B[a]P-Modified DNA Oligonucleotides

Custom DNA oligonucleotides were purchased from Eurofins MWG Operon and purified by high-performance liquid chromatography (HPLC) using a reverse phase C18 column (all sequences in Supplementary Table S1). Template oligos were Cy3-labeled at an amino-modified C6-dT with a Cy3 NHS ester (GE healthcare) as described^{54 (link)}. DNA purity was assessed by MALDI-TOF MS.
B[a]P-modified template was prepared as described^{55 (link)} (Supplementary reaction scheme S1). Briefly, a racemic mixture of (±)-anti-B[a]PDE (National Cancer Institute Chemical Reference Standard Repository, Kansas City, MO) was incubated with an 11-mer containing a single dG. The reaction yields four isomeric products that were separated by reverse phase HPLC. The selected B[a]P-modified 11-mer was ligated to a Cy3-labeled 15-mer using T4 DNA ligase and the final product (26-mer) was purified by reverse phase HPLC with a heated column. The 26-mer unmodified template and B[a]P modified template were heat annealed to the appropriate primer depending on the experiment.