PMC

Structures of four of the dinucleoside tetraphosphates that were synthesized.

Schematics for synthesis of ddNp₄ddNs. A 10 μM concentration of DNA P/T was incubated with a 3.2 mM concentration of the ddNTP (a small portion of which was α-³³P labeled) that is complementary to the first single-stranded position on the template, 1 μM multinucleoside-resistant HIV-1 RT (HIV-1 RT^MDR), and 0.5 units of inorganic pyrophosphatase. Incorporation of the 2′,3′-dideoxynucleoside monophosphate into the primer terminus leads to chain termination. The PP_i generated by the incorporation was cleaved by pyrophosphatase, and the chain terminator was transferred from the blocked DNA to ddNTP, leading to formation of the dinucleoside polyphosphate ddNp₄ddN and unblocked P/T available for another round of synthesis.

Inhibition of DNA-dependent DNA synthesis through incorporation of ddGMP from Ap₄ddG by RT^WT, RT^AZT, or RT^MDR. (A) M13 single-stranded DNA annealed to a DNA primer (2.5 nM) was incubated with HIV-1 RT^WT or HIV-1 RT^AZT (100 nM), a 2.5 μM concentration of each dNTP, 50 μCi of [α-³²P]dGTP, and the indicated concentrations of Ap₄ddG for 30 min at 37°C. The samples were separated by 20% urea-polyacrylamide gel electrophoresis. (B) Percent inhibition of [³²P]dGMP incorporation by Ap₄ddG was determined as described in Materials and Methods. A summary of IC₅₀s derived from several experiments is given in Table .

Products formed during incorporation of ddGMP from doubly labeled Ap₄ddG. A 10 μM concentration of unlabeled WL50-33C/L32 template/primer was incubated with 2 μM HIV-1 RT^MDR and 200 μM Ap₄ddG (A-P-P-³²P-³³P-ddG) for the indicated times at 37°C, followed by heat inactivation of the RT (5 min at 95°C). Some samples were then treated with 4 U of CIP (+CIP) for 30 min, followed by heat inactivation (5 min at 95°C) of the CIP. The reaction products were separated by 20% urea-polyacrylamide gel electrophoresis, and the radioactivity was visualized by phosphorimaging. Standards for free phosphate and PP_i were generated by incubating [γ-³²P]ATP with CIP or snake venom phosphodiesterase (SVPD), respectively.

Synthesis and purification of ddNp₄ddNs. (A) A 10 μM concentration of one of four different DNA P/Ts (see Materials and Methods for sequences) was incubated with HIV-1 RT^MDR, inorganic pyrophosphatase, and 3.2 mM ddATP (lanes 1 and 5), ddCTP (lanes 2 and 6), ddGTP (lanes 3 and 7), or ddTTP (lanes 4 and 8) for 7 days at 37°C. Following heat inactivation of the RT, the reaction mixture was incubated with CIP (lanes 5 to 8). (B) The CIP-treated reaction mixture shown in lane 5 in panel A was loaded onto an anion exchange column and eluted with a 10 to 1,000 mM gradient of triethylammonium bicarbonate (TEAB) buffer, pH 8.5. Aliquots of reaction mixtures or selected column fractions were separated by electrophoresis through 20% urea-polyacrylamide gel electrophoresis.

Inhibition of DNA synthesis and formation of stable complexes with RT^WT and RT^AZT by ddGTP or ddGp₄ddG. (A) M13 single-stranded DNA annealed to a cDNA primer (2.5 nM) was incubated with HIV-1 RT^AZT (100 nM), a 2.5 μM concentration of each dNTP, 50 μCi of [α-³²P]dGTP, and the indicated concentration of ddGTP or ddGp₄ddG that was either untreated (−) or had been pretreated with CIP (+) for 30 min at 37°C. The samples were separated by 20% urea-polyacrylamide gel electrophoresis. (B) Electrophoretic mobility shift assay for the ability of RT^WT or RT^AZT to form a stable dead-end complex (DEC) with ddAMP-terminated primer/template. Enzyme was incubated with WL50-33C/L31-[³²P]ddAMP template/primer in the presence of the indicated concentration of either ddGTP or ddGp₄ddG, and stable complexes were separated from free DNA by nondenaturing electrophoresis as described in Materials and Methods. K_D values were determined from plots of the percentage of total radioactivity in complex versus substrate concentration. DNA P/T sequence near the primer terminus is given at the bottom of the panel.