Enzymatic assay for quantification of deoxynucleoside triphosphates in human cells exposed to antiretroviral 2',3'-dideoxynucleosides.
Quantification of intracellular 2'-deoxynucleoside-5'-triphosphates (dNTPs) is of importance in studies of antiretroviral 2',3'-dideoxynucleoside analogs (ddNs) and a highly sensitive enzymatic assay for dNTPs has frequently been used for this purpose. However, the susceptibility of the assay to interference from the corresponding substrate analogs, ddNTPs, is still undefined. Ideally, DNA polymerases used in the assay should meet at least two criteria: (i) high fidelity to the template even in the presence of ddNTPs and (ii) low affinity for ddNTPs. None of the currently used exonuclease-free Klenow and Sequenase enzymes met both criteria. However, Sequenase had higher fidelity to the template than did the Klenow enzyme in the presence of pyrimidine-ddNTPs, and its reaction followed first order kinetics. We have, therefore relying primarily on Sequenase, designed a dNTP proportional reduction assay to correct the ddN-induced deviation in the enzymatic assay. With the use of high-fidelity exonuclease-free DNA polymerase and the application of correction factors, we now can accurately quantify dNTPs with a minimum detection limit as low as 0.1 pmol, using as few as 1 x 10(4) peripheral blood mononuclear cells. The method described should be useful in the study and development of antiretroviral ddNs.
Gao
W Y
WY
Experimental Retrovirology Section, National Cancer Institute, Bethesda, Maryland 20892.
Johns
D G
DG
Mitsuya
H
H
D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000032:analysis*; D015224:Dideoxynucleosides / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012190:Retroviridae / Q000187:drug effects*
D000998:Antiviral Agents; D003854:Deoxyribonucleotides; D015224:Dideoxynucleosides; D009838:Oligodeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase