| Assay Method Information | |
| | Inhibition of HIV Reverse Transcriptase |
| Description: | Recombinant full length HIV-1 Reverse Transcriptase (HIVrt) was purchased from Abcam, catalog #ab63979. The last 385 nucleotide region of the HCV anti-genome complementary to the 5′ untranslated region (c5′UTR) was synthesized using T7 RNA polymerase Megascript kit from Ambion (Cat #AM1333). A DNA oligo served as an internal initiation primer and was purchased from IDT. Unless otherwise specified, reaction samples consisted of 20 nM c5′UTR RNA, 100 nM DNA primer, and 1 nM HIVrt, mixed together in a buffer containing 50 mM Tris pH 7.5, 100 mM KCl, 4 mM dithiothreitol (DTT), and 12.5 mM MgCl2. Reactions were initiated at 30° C. by adding 0.1 μM adenosine triphosphate (dATP), 0.1 μM cytosine triphosphate (dCTP), 1 μM guanosine triphosphate (dGTP), and 0.32 μM 3H-thymidine triphosphate (3H-TTP), in a final volume of 50 μL. After 40-mins incubation, the reaction was terminated by adding 60 μl of chilled 20% (w/v) trichloroacetic acid with 500 μM ATP to precipitate nucleic acids. After incubation at 4° C. for 1 h, the sample underwent filtration on a multiscreen BV 1.2-μm 96-well plate (Millipore). 40 μL Microscint-20 (Perkin Elmer) was added to the well and the counts in the sample were determined by a Trilux Microbeta microplate scintillation reader (Wallac).All data were analyzed with GraphPad Prism. The compound concentration at which the enzyme-catalyzed rate was reduced by 50% (IC50) was calculated by fitting the data to the equation Y=% Min+(% Max−% Min)/(1+X/IC50), where Y corresponds to the percent relative enzyme activity, % Min is the residual relative activity at saturating compound concentration, % Max is the relative maximum enzyme activity, and X corresponds to the compound concentration. The Ki was calculated using the Cheng-Prusoff equation assuming competitive inhibition relative to natural dNTP incorporation: Ki=IC50/(1+[dNTP]/Km), where [dNTP] is the concentration of natural dNTP and Km is the apparent Km for dNTP. The standard HIVrt RNA-dependent DNA polymerization (RdDp) assay was used to determine the IC50 values. |
| Affinity data for this assay | |
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