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| | Endonuclease Assay |
| Description: | The PAN domain has been shown to cleave ssRNA as well as ssDNA. To demonstrate the inhibition of endonuclease cleavage by PAN, a high throughput assay was developed (U.S. patent application Ser. No. 13/554,709). A TaqMan-like oligonucleotide was used containing a 6-carboxy-fluorescein (FAM) fluorophore at the 5′-end followed by 19 nucleotides and a minor groove binding non-fluorescent quencher (MGBNFQ, Applied Biosystems) at the 3′-end. When excited by light at a wavelength of 488 nm, MGBNFQ quenches the fluorescence of FAM via fluorescence resonance energy transfer. If the endonuclease cleaves the oligonucleotide, the quencher is no longer coupled to the fluorophore, and therefore, FAM fluoresces. This assay can be performed in a high-throughput (e.g. 96 well plate) format. The assay can be used to evaluate the inhibitory characteristics of compounds that are found to bind PAN and to screen libraries of drug-like compounds. The assay uses the probe 6FAM-TGGCAATATCAGCTCCACA-MGBNFQ. The assay can be performed in a 40 μl reaction volume with 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgSO4, 0.05 mM MnSO4, 1 mM DTT, 0.75 mM CHAPS, 50 nM probe, and 25 nM endonuclease. The reaction mixture is set up as a master mix with the buffer, probe, and protein on ice. The inhibitor is then added to a maximum DMSO concentration of 2.5% (v/v) and serial dilutions are made on ice. Varioskan Fluorometer (Thermo Scientific), set to an excitation of 488 nm and emission of 518 nm, is used to measure the fluorescence of the samples at 37 degrees Celsius. Fluorescence is measured at various time points (5, 120, and 240 minutes) during the 37 degrees Celsius incubation. Activity/inhibition is calculated based on the change in fluorescence over time using Prism Graphpad non-linear regression analysis. |
| Affinity data for this assay | |
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