7000 Nucleic Acids Research, 2008, Vol. 36, No. 22 form stable nA-T and A-sT base pairs but together func- tion as a mismatch (10). The dNTPs of these bases are readily incorporated into DNA (11). We recently reported that 7-ethyl-7-deazaguanine (EtcG) and N4-ethylcytosine (EtC) together with the corresponding methyl derivatives provide a route to pseudo-complementary DNA (12). These analogs, which are weakly to moderately destabiliz- ing when base paired to C or G, interact poorly with one another. DNA substituted with nA, sT, EtcG and EtC is structure-free but is relatively hydrophobic and challen- ging to synthesize. Here we examine several highly desta- bilizing analogs of G and C for one that can confer pseudo-complementarity to DNA in the presence of nA and sT. Several of these analogs have pharmacological signiﬁ- cance including 6-thioguanine (sG) (13), 2-pyrimidinone (P) (14), 6-methylfuranopyrimidinone (MefP) (15) and 5-nitrocytosine (NitroC) (16). sG is an anticancer agent commonly used for treating leukemia. P is a potent inhi- bitor of DNA methylation and may have application in the epigenetic therapy of cancer. Both NitroC and certain derivatives of MefP possess antiviral properties. While sG and P must be incorporated into DNA to exhibit activity this may not be the case of MefP and NitroC. The struc- ture-activity relationships reported here provide an oppor- tunity to compare the enzymatic uptake and base pairing properties of these bases. EXPERIMENTAL PROCEDURES Materials Unmodiﬁed oligodeoxynucleotides were purchased from IDT (Coralville, IA) and locked nucleic acids were a gift from Sigma Proligo (Paris, France). Modiﬁed oligodeoxy- nucleotides that contained H or sG were synthesized using phosphoramidites from Glen Research (Sterling, VA). Vent (exo-) DNA polymerase was obtained from New England Biolabs (Ipswich, MA). Exo- T7 DNA polymer- ase (Sequenase) was purchased from USB (Cleveland, OH). The N62D mutant of f29 DNA polymerase was obtained from M. Salas (Instituto de Biologia Molecular ‘Eladio Vin˜ uela’, Madrid, Spain). Deoxynucleoside triphosphates dNTPs of nA, sT, hypoxanthine (H), sG, 2-thiocytosine (sC), 7-deazaguanine (cG) and P are commercially avail- able from TriLink Biotechnologies (San Diego, CA). Deoxynucleosides of 7-deazahypoxanthine (cH) and 6-methylfuranopyrimidinone (MefP) were purchased, respectively, from ChemGenes (Wilmington, MA) and Berry & Associates (Dexter, MI). Deoxynucleosides of 7-iodo-7-deazahypoxanthine (IcH), 7-nitro-7-deazahypox- anthine (NitrocH), 7-nitro-7-deazaguanine (NitrocG) and 5-nitrocytosine (NitroC) were synthesized as described in Supplementary Data. Nucleosides were phosphorylated by stirring 0.3–0.4 mmol quantities with 1.5–3 equivalents of POCl3 in 2–3 ml of dry trimethylphosphate for several hours at 08C. Reactions were terminated by adding 5 equivalents of tributylammonium pyrophosphate (0.5 M in acetonitrile) and 4 equivalents of tributylamine with vigorous stirring. After 5 min 5–10 ml of 1 M triethyl- ammonium bicarbonate (TEAB) was added with stirring for an additional 30 min. Each dNTP was isolated and puriﬁed by a combination of anion exchange (water–1 M TEAB) and reverse phase (100 mM TEAB–acetonitrile) chromatography. The isolated triethylammonium salt of the dNTP was converted into the Li salt by precipitation from 2% lithium perchlorate in acetone. Isolated yields were 15–30%. The synthesis of 7-nitro-7-deaza-20-deoxy- guanosine 50-triphosphate required the presence of 1,8-bis(dimethyl-amino)naphthalene (Proton Sponge, 2 equiv.) to prevent degradation of the nucleoside base (17). Primer extension Most primer extension reactions were conducted at 308C for 30 min in the presence of 0.6 mM 32P end-labeled primer, 1.2 mM template, 200 mM each dNTP, and 0.05 mg/ml f29 N62D DNA polymerase in 40 mM Tris– HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 4 mM (NH4)2SO4 (12). Reactions that contained T7 instead of f29 DNA polymerase were carried out as previously described (11). Labeled DNA products were resolved by electrophoresis of reaction aliquots through a 7 M urea–12% polyacrylamide gel. Since the product of primer extension diﬀered in length from its template, denaturing gel electrophoresis aﬀorded pure single- stranded target for use in hybridization reactions. Primer extension products with a hairpin conﬁguration were not gel puriﬁed prior to use as hybridization targets. Rather, these reactions were diluted 15-fold with water and heated 2 min at 958C in the presence of an oligonucleotide (0.4 mM) that was complementary to the 30 overhang of the template. Upon quick cooling in an ice bath the tem- plate and product strands collapsed into separate hairpins. The oligonucleotide prevented reannealing of template and product hairpins by blocking hybridization between their complementary single-stranded tails. Hybridization studies The hybridization assays used here have also been described in previous publications (11,18). Brieﬂy, hybri- dizations were conducted in 5 mM MgCl2, 25 mM NaCl, 20 mM HEPES, pH 7.5 using synthetic oligonucleotides as probes and end-labeled primer extension products or che- mically synthesized oligomers as targets. Apparent melting temperatures of hairpins were determined by incubating a trace amount of labeled hairpin with 5 mM of a probe equal in length and complementary to one arm of the hairpin. Incubations were conducted over a wide range of temperatures using 58C increments. After 5 min each reaction aliquot was quenched in an ice bath and analyzed by gel mobility shift through a 12% non-denaturing gel run in the presence of 5 mM MgCl2 in a cold room. A somewhat diﬀerent protocol was used to determine the apparent melting temperatures of intermolecular Watson–Crick hybrids. Probes (1 mM) were hybridized to a trace amount of radiolabeled single-stranded target by raising the temperature to 908C and then lowering it to 48C in a thermocycler. Aliquots of the hybrids were again incubated over a wide temperature range. After 5 min