3′‐Exonuclease resistance of DNA oligodeoxynucleotides containing O6‐[4‐oxo‐4‐(3‐pyridyl)butyl]guanine

Soobong Park 2 Mahadevan Seetharaman 0 1 Alexis Ogdie 2 David Ferguson 0 1 Natalia Tretyakova 1 2 0 Minnesota Supercomputing Institute, University of Minnesota , Minneapolis, MN 55455, USA 1 Department of Medicinal Chemistry, University of Minnesota School of Pharmacy 2 University of Minnesota Cancer Center Tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a chemical carcinogen thought to be involved in the initiation of lung cancer in smokers. NNK is metabolically activated to methylating and pyridyloxobutylating species that form promutagenic adducts with DNA nucleobases, e.g. O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-POB-dG). O6-POB-dG is a strongly mispairing DNA lesion capable of inducing both GfiA and GfiT base changes, suggesting its importance in NNK mutagenesis and carcinogenesis. Our earlier investigations have identified the ability of O6-POBdG to hinder DNA digestion by snake venom phosphodiesterase (SVPDE), a 3-exonuclease commonly used for DNA ladder sequencing and as a model enzyme to test nuclease sensitivity of anti-sense oligonucleotide drugs. We now extend our investigation to three other enzymes possessing 3-exonuclease activity: bacteriophage T4 DNA polymerase, Escherichia coli DNA polymerase I, and E.coli exonuclease III. Our results indicate that, unlike SVPDE, 3-exonuclease activities of these three enzymes are not blocked by O6-POB-dG lesion. Conformational analysis and molecular dynamics simulations of DNA containing O6-POB-dG suggest that the observed resistance of the O6-POB-dG lesion to SVPDE-catalyzed hydrolysis may result from the structural changes in the DNA strand induced by the O6-POB group, including C3-endo sugar puckering and the loss of stacking interaction between the pyridyloxobutylated guanine and its flanking bases. In contrast, O6-methylguanine lesion used as a control does not induce similar structural changes in DNA and does not prevent its digestion by SVPDE. - Cigarette smoking is a well known risk factor for lung cancer. The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone, (NNK; I in Scheme 1) is thought to be involved in the induction of lung cancer in smokers (1,2). Indeed, NNK has been reported to be metabolically activated to [4-(3-pyridyl)-4-oxobutyl]diazohydroxides, II and methyldiazohydroxide, III (Scheme 1), which pyridyloxobutylate and methylate guanines in DNA, respectively (24). If not repaired before DNA replication, NNK-induced DNA lesions can be misread by DNA polymerases, resulting in heritable mutations and the initiation of cancer (Scheme 1). We and others have developed methods for mapping carcinogen-induced nucleobase lesions within DNA sequences by mass spectral analysis of controlled exonuclease digests (5,6). Exonuclease enzymes sequentially remove mononucleotides in either the 5fi3 or the 3fi5 direction. Since time controlled exonuclease digests contain a mixture of DNA fragments differing from each other by one or more mononucleotides (DNA ladders), mass differences between adjacent peaks in the resulting mass spectra correspond to individual nucleotides, making it possible to determine the complete DNA sequence (5,6). The 16mer d(AACAGCCATAT[O6-POB-dG]GCCC) was digested with 5fi3 and 3fi5 exonucleases in order to confirm the position of O6-[4oxo-4-(3-pyridyl)butyl]guanine (O6-POB-dG) in the sequence (5). Interestingly, while bovine spleen phosphodiesterase (5exonuclease) was able to remove pyridyloxobutylated guanine nucleotide (O6-POB-dG, M = 477.2), snake venom phosphodiesterase (SVPDE, 3-exonuclease) was incapable of bypassing the lesion (5). The ability of O6-POB-dG to block SVPDE-catalyzed DNA hydrolysis is of potential interest because of the continuing effort to develop nuclease-resistant antisense oligodeoxynucleotides (ODNs). 3-Exonucleases have been shown to be primarily responsible for enzymatic degradation of therapeutic ODNs in serum-containing medium and in various cell lines (79). The antisense activity of phosphodiester ODNs in cells is primarily determined by their resistance to exonucleolytic degradation (10). Monia et al. (10) have proposed that SVPDE can be used as a model nuclease in experiments that examine nuclease sensitivity of antisense cellular nuclease responsible for degradation of antisense oligonucleotides. Many chemically modified antisense ODNs have been designed to confer nuclease resistance while preserving the antisense activity, including the introduction of cationic groups (9,11), bulky tricyclic base moieties (12), phosphothiolate backbone linkages and 2-substituted sugars (10), self-forming 3 hairpins (13), 2-O,4-C-ethylene linked nucleosides (14), 3-conjugates (8), ODNs of reversed polarity (2,5) (15), and a-anomeric oligonucleotides (16). Although SVPDE alone is commonly used to assess the exonucleolytic stability of chemically modified ODNs, structural differences between the active site geometries of different nucleases may render O6-POB-dG-containing DNA less resistant towards physiologically relevant 3exonucleases. We thus examined the ability of three other common 3-exonucleases [E.coli exonuclease III, E.coli DNA polymerase I (KF), and T4 DNA polymerase] to hydrolyze O6-POB-dG-containing DNA. Furthermore, torsional analysis and molecular modeling simulations of O6-POB-dGcontaining DNA were performed in an attempt to uncover the structural basis for the observed exonuclease resistance of pyridyloxobutylated DNA. MATERIALS AND METHODS 3-Hydroxypicolinic acid (HPA) was purchased from Bruker Daltonics (Billerica, MA). Bio-gel P-6 cartridges were obtained from Bio-Rad Laboratories (Hercules, CA). SVPDE and E.coli DNA polymerase I were purchased from Worthington Biochemicals (Lakewood, NJ). T4 DNA polymerase was from Invitrogen Life Technologies (Carlsbad, CA). Escherichia coli exonuclease III was obtained from Epicentre Technologies (Madison, WI), and the Klenow fragment of E.coli DNA polymerase I was purchased from Sigma (St Louis, MO). O6-Me-dG phosphoamidite was obtained from Glen Research (Sterling, VA). Parafilm was obtained from American National Can (Greenwich, CT). Cation exchange beads in ammonium form were purchased from Applied Biosystems (Foster City, CA). Water was purified with a Milli-Q ultrapure water filtration system from Millipore (Bedford, MA). The DNA oligomer sequences used in our study are listed in Table 1. The O6-POB-dG-containing ODN d(AACAGCCATAT[O6-POB-G]GCCC) was kindly provided by Professor Lisa Peterson (University of Minnesota Cancer Center). The hexadecamer of the same sequence with O6-Me-dG in place of O6-POB-dG and the complemetary DNA strands were prepared by standard phosphoramidite chemistry at Microchemical Facility at University of Minnesota. The O6Me-dG phosphoramidite was obtained from Glen Research. The synthetic DNA oligomer was deprotected by a manufacturer-supplied method, and HPLC purified as described elsewhere (17). The concentra (...truncated)