Synthesis of Oligonucleotides Containing the N²-Deoxyguanosine Adduct of the Dietary Carcinogen 2-Amino-3-methylimidazo[4,5-f]quinoline

Abstract

Abstract2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine formed in all cooked meats. IQ has been found to be a potent inducer of frameshift mutations in bacteria and carcinogenic in laboratory animals. Upon metabolic activation, IQ forms covalent adducts at the C8-and N 2 -positions of deoxyguanosine with a relative ratio of up to ~4:1. We have previously incorporated the major dGuo-C8-IQ adduct into oligonucleotides through the corresponding phosphoramidite reagent. We report here the sequence-specific synthesis of oligonucleotides containing the minor dGuo-N 2 -IQ adduct. Thermal melting analysis revealed that the dGuo-N 2 -IQ adduct significantly destabilizes duplex DNA.

Base-displaced intercalation of the 2-amino-3-methylimidazo[4,5-f]quinolone N2-dG adduct in the NarI DNA recognition sequence

“…The synthesis of this adduct into oligodeoxynucleotides (67) has allowed the conformation of the N 2 -dG-IQ adduct at the G 3 position of this sequence to be determined. This is a hot spot for two-base frameshift deletions in bacterial mutagenesis assays (68–71,73,74).…”

“…The N 2 -dG-IQ-adducted oligodeoxynucleotide 5′-d(CTCGGCXCCATC)-3′ was synthesized as described ( 67 ). The complement strand 5′-d(GATGGCGCCGAG)-3′ was synthesized by Midland Certified Reagents Co. (Midland, TX, USA) and purified by anion exchange chromatography.…”

Base-Displaced Intercalated Conformation of the 2-Amino-3-methylimidazo[4,5-f]quinoline N2-dG DNA Adduct Positioned at the Nonreiterated G1 in the NarI Restriction Site

“…The N 2 -dG-IQ adduct was incorporated into 5′-d(CTC X GCGCCATC)-3′ using automated solid-phase synthesis. 63 It was located at position X 4 , corresponding to position G 1 in the Nar I sequence (Chart 1 ). The modified oligodeoxynucleotide was purified by C18 reverse-phase HPLC and characterized by MALDI-TOF mass spectrometry in negative ion mode (modified strand m / z obsvd, 3779; calcd, 3778; complementary strand m / z obsvd, 3712; calcd, 3711).…”

“…26 It may play a significant role in cancer etiology. 18 , 19 Polymerase bypass studies conducted in vitro have revealed that the consequences of replication past the N 2 -dG-IQ adduct differ when the lesion was incorporated 63 , 72 at position G 1 vs G 3 of the Nar I restriction endonuclease sequence, 5′-d(G 1 G 2 CG 3 CC)-3′. 66 − 71 Two-base deletions were produced by hpol η when the N 2 -dG-IQ adduct was located at the G 3 position, but when this lesion was located at the G 1 position, error-free incorporation of dCTP occurred but further replication stalled.…”

“…The synthesis of the N 2 -dG-IQ-adducted oligodeoxynucleotide, 5′-d(CTC X GCGCCATC)-3′, has been described. 63 The oligodeoxynucleotide 5′-d(GATGGCGCCGAG)-3′, purified by anion exchange chromatography, was obtained from the Midland Certified Reagent Company (Midland, TX). The oligodeoxynucleotides were purified by HPLC using an acetonitrile gradient from 5 to 12% over 35 min in ammonium formate (pH 7) with a base-deactivated C18 Supesilco LS-18-DB column (Sigma-Aldrich, St. Louis, MO).…”