RESUMO
A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)].
Assuntos
Reação em Cadeia da Polimerase/métodos , Telomerase/análise , Telômero/metabolismo , Extratos Celulares , Ácidos Cólicos/farmacologia , DNA/isolamento & purificação , DNA/metabolismo , Primers do DNA/metabolismo , Detergentes/farmacologia , Etanolaminas/farmacologia , Células HL-60 , Células HeLa , Humanos , Cinética , Oligonucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Sensibilidade e Especificidade , TemperaturaRESUMO
Oligodeoxycytidylates were converted to s4dUMP-containing oligomers by treatment with liquid H2S. The inhibitory potency of the modified oligonucleotides on human immunodeficiency virus type 1 reverse transcriptase depended on the chain length and on the percentage of modification. The most potent reverse transcriptase inhibitor was (s4dU)35. The inhibitory pattern was competitive, when either poly(A) x (dT)16 or poly(C) x (dG)l6 was used as template-primer (variable substrate), suggesting that the free enzyme interacts with (s4dU)35. The Ki values were 3.0 and 2.2 nM in the presence of poly(A) x (dT)16 and poly(C) x (dG)16, respectively.
Assuntos
Nucleotídeos de Desoxiuracil/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Cromatografia Líquida de Alta Pressão , Primers do DNA , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Enxofre/químicaRESUMO
Partially 5-thiolated polyuridylic acid (poly(U60,hs5U40)) is shown to be a potent inhibitor of Moloney murine leukemia virus Reverse Transcriptase (M-MuLV RT). The pattern of this inhibition is competitive, when either poly(A).(dT)16 or poly(C).(dG)16 as template-primer (variable substrate) are used, suggesting that the free enzyme interacts with the modified polynucleotide. Km and Ki values of 25 microM and 11 nM, respectively, were obtained in the presence of poly(A).(dT)16. The Ki value determined in the presence of poly(C).(dG)16 was 31 nM (Km = 22 microM). Inhibitory activities of the 5-thiolated oligouridylic acids, prepared from the polymer, depend on the chain-length. While the 30-mer showed the same activity as the intact polynucleotide, shorter oligonucleotide proved to be less active.
