The deoxyribooligonucleotide 5′-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(−)-anti-BPDE)], to produce the modified oligonucleotides 5′-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (−)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3′-OH terminus to the 5′-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (−)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5′ → 3′ direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5′-d(…T-G*…)-3′ bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (−)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3′-side of the modified G [5′-d(…G*-T…)-3′] is most resistant to digestion by both enzymes. It is concluded that in single-stranded oligonucleotides the pyrenyl residues point toward the 5′-end, while in (−)-trans adducts they point toward the 3′-end of the modified strands, paralleling the orientations recently found by 2D NMR techniques in analogous (+)-trans- and (−)-trans-BPDE-oligonucleotide duplexes [de los Santos et al. (1992) Biochemistry 31, 5245–5252]. Overall rates of enzyme digestion are more hindered whenever the bulky pyrenyl residue points in a direction opposing the progress of hydrolysis and are more facile when the pyrenyl ring points along the direction (3′ → 5′ or 5′ → 3′) of exonuclease digestion. Identification of exonuclease digestion stall sites in single-stranded oligonucleotides modified with bulky adducts may prove useful for deducing the orientations of these covalently bound residues relative to the modified base and the 5′-3′ strand polarity.
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