title: Development of nonradioactive microtiter plate assays for nuclease activity.
abstract: We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.
authors.affiliation: Laboratoire d'Ingénierie des Anticorps, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. mouratou@pasteur.fr
authors.forename: Barbara
authors.initials: B
authors.lastname: Mouratou
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rouyre
authors.forename: Serge
authors.initials: S
authors.lastname: Pauillac
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Guesdon
mesh_terms: D001312:Aurintricarboxylic Acid / Q000737:chemistry; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000032:analysis / Q000378:metabolism*; D004076:Digoxigenin / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008863:Microspheres; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000032:analysis / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry; D013997:Time Factors
chemical_list: D009841:Oligonucleotides; D001312:Aurintricarboxylic Acid; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D004492:Edetic Acid; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004076:Digoxigenin