Synthesis of DNA Oligodeoxynucleotides Containing Site‐Specific 1,3‐Butadiene‐ Deoxyadenosine Lesions

Abstract

Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis.

Base Excision Repair of N6-Deoxyadenosine Adducts of 1,3-Butadiene

“…28,29 DNA strands containing ( R,R )- N 6 -(2-hydroxy-3,4-epoxybutan-1-yl)-dA were converted to the corresponding ( R,S )-1, N 6 -HMHP-dA containing strands via intramolecular nucleophilic substitution in an aqueous solution. 28,29 These reactions were conducted on solid support in order to maximize the yield of the final product. All synthetic oligodeoxynucleotides were purified by HPLC to achieve > 98% purity and characterized by liquid chromatography-mass spectrometry (Table 1, Supplement S-1).…”

“…The corresponding oligomers containing ( S )- N 6 -HB-dA and (R,R) - N 6 , N 6 -DHB-dA adducts were prepared via nucleophilic aromatic substitution of 6-chloropurine-containing DNA on solid support with ( S )- N -Fmoc-1-aminobut-3-en-2-ol and ( R,R ) - pyrrolidine-3,4-diol, respectively (Scheme 2). 29 Adduct-containing oligodeoxynucleotides were cleaved off solid support using 0.1 M NaOH for three days at room temperature. 29 DNA strands containing 5-fluoro-dU, 8-oxo-dG and 1, N 6 -etheno-dA at position X (positive controls for BER experiments) were prepared by solid phase synthesis using commercially available phosphoramidites (Glen Research, Sterling, VA).…”

“…29 Adduct-containing oligodeoxynucleotides were cleaved off solid support using 0.1 M NaOH for three days at room temperature. 29 DNA strands containing 5-fluoro-dU, 8-oxo-dG and 1, N 6 -etheno-dA at position X (positive controls for BER experiments) were prepared by solid phase synthesis using commercially available phosphoramidites (Glen Research, Sterling, VA). The modified nucleotides were added using an offline manual coupling protocol.…”

“…Synthetic oligodeoxynucleotides containing site-specific and stereospecific ( S )- N 6 -HB-dA, ( R , S )-1, N 6 -HMHP-dA, and ( R , R )- N 6 , N 6 -DHB-dA lesions were prepared by the postoligomerization methodology developed in our laboratory. − Briefly, ( R , S )-1, N 6 -HMHP-dA-adducted DNA strands were prepared by coupling ( R , R )- N -Fmoc-1-amino-2-hydroxy-3,4-epoxybutane with the oligomers containing site-specific 6-chloropurine at position X . The resulting ( R , R )- N 6 -(2-hydroxy-3,4-epoxybut-1-yl)­adenine [( R , R )-HEB-dA]-containing oligodeoxynucleotides were isolated by high-performance liquid chromatography (HPLC) and subjected to cyclization in water to afford the corresponding ( R , S )-1, N 6 -HMHP-dA strands.…”

Synthesis and Characterization of Site‐Specific O6‐Alkylguanine DNA‐Alkyl Transferase‐Oligonucleotide Crosslinks

“…Post-oligomerization can be done in solution or at the CPG bead level (Wickramaratne, Seiler, & Tretyakova, 2015). For the synthesis of N 6 -propargyl-dA template, the post-oligomerization reaction was carried out in solution as described in Basic Protocol 2.…”