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The oligonucleotide 6-Fam-dArUdAdA-BHQ1 is used as a substrate for a convenient proof of RNases. In inactivated state the quencher causes full extinction of the fluorescence of fluorescein. After digestion by the RNase, however, quencher and fluorophore are separated from each other and thus the fluorescence of the 6-Fam reporter can be detected at 520 nm.
|Increase of fluorescence induced by digestion of the substrate (200 pM RNase A, 100 nM RNase substrats, 100 mM MES buffer, pH= 6.0, 100 mM NaCl), detection at 515 nm, excitation at 490 nm
||Fluorescene spectrum of the cleaved substrate|
1. Hypersensitive substrate for ribonucleases. Kelemen BR, Klink TA, Behlke MA, Eubanks SR, Leland PA, Raines RT; Nuc. Acids Chem. (1999), 27, 3696-3701.
2. A Ribonuclease Variant with Low Catalytic Activity but High Cytotoxicity. Bretscher LE, Abel RL, Raines RT. J. Biol. Chem. (2000), 275.