Assays for nucleic acid modifying enzymes
View Patent ↗Methods and compositions are provided for assaying for enzymes capable of releasing an enzyme donor fragment (ED) conjugated to a nucleic acid strand bonded to a surface. Conveniently, the beads are magnetic allowing segregation of the beads during the determination. Upon addition of enzyme acceptor fragment (EA) and substrate to the assay mixture, the method allows for discrimination between ED free in solution and ED bound to the bead. The complexing of ED and EA provides an active β-galactosidase enzyme. The method permits the assay of any substance involved in a pathway that can result in a reaction releasing the ED.
1. A method for detecting the effect of a test compound being tested for effect on nuclease activity, said method comprising:
incubating in an assay medium said test compound, a nuclease which is either an endonuclease or exonuclease V, and an enzyme donor fragment (ED)-reagent for sufficient time for reaction of said ED-reagent and said nuclease, said ED-reagent comprising ED linked through an oligonucleotide strand of not more than 60 nucleotides to a magnetic bead,
said reaction resulting in separating said ED from said magnetic bead;
adding enzyme acceptor (EA) and a β-galactosidase substrate that allows for determination of an amount of turnover of said substrate;
wherein said ED and EA are fragments of β-galactosidase, and said separating of said ED from said magnetic bead results in complexing of said ED and said EA to form an active β-galactosidase enzyme, and said ED reagent has substantially reduced ability to complex with said EA as compared to ED separated from said bead, thereby resulting in reduced background; and
determining the amount of turnover of active β-galactosidase enzyme formed from said ED and said EA as a measure of the effect of said test compound on said nuclease activity.
2. The method according to claim 1 , wherein said test compound is derived from at least one of a source of mRNA, a source of DNA, or a source of a nuclease activating enzyme, wherein said turnover is related to said mRNA, said DNA or said nuclease activating enzyme, respectively.
3. The method according to claim 2 wherein said source of DNA provides DNA complementary to said oligonucleotide strand.
4. The method according to claim 1 , wherein unmodified beads are included in said assay medium prior to said determining.
5. The method according to claim 1 , wherein said ED is bonded to a single stranded oligonucleotide strand bonded to said bead.
6. The method according to claim 1 , wherein said ED is bonded to a second oligonucleotide strand hybridized to a first oligonucleotide strand bonded to said bead, whereby said ED is linked through an oligonucleotide strand to said magnetic bead.
7. The method according to claim 1 , wherein prior to said adding, said magnetic bead is segregated by means of a magnetic field.
8. The method of claim 6 wherein said second oligonucleotide strand comprises synthetic nucleotides.
9. The method according to claim 1 wherein said endonuclease is a restriction enzyme.