Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-07-31
2000-08-08
Guzo, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 9131, C12Q 168, C12P 1934
Patent
active
061000280
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a novel sensitive assay to detect and quantify the presence of an enzyme which acts to produce a single-stranded oligonucleotide product, or to inhibitors of such enzyme.
BACKGROUND OF THE INVENTION
Enzymes which use polynucleotides as substrates are often fundamental to the biochemical function of many organisms and viruses. This group of enzymes includes, for example, endonucleases, exonucleases and ribozymes. A number of enzymes act on substrates to give single-stranded oligonucleotide products. For example, the influenza virus endonuclease cleaves capped host cell transcripts 10 to 13 bases from the 5' end. Sequence specific RNA endonucleases are known; for example the 2-5A-dependent RNase found in higher animals functions to cleave single-stranded regions of RNA 3' of UpNp dimers, with a preference for UU and UA sequences. In addition, ribozymes are known to cleave single-stranded RNAs at specific recognition sequences.
While it is recognized that inhibitors or activators of these enzymes might be new classes of therapeutic or preventative compounds, particularly against viral diseases, identification of such compounds has been hampered by the lack of a convenient assay system.
An ideal enzyme assay system should have: a) high throughput; b) the ability to distinguish enzyme-catalyzed cleavage from nonspecific nucleotide cleavage; and c) high sensitivity. Previous nucleotide cleavage assays involved the use of polyacrylamide gel electrophoresis to separate product from substrate (Plotch et al., 1981 Cell 23:847-858) which is not convenient for processing large numbers of samples.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a novel, accurate, sensitive, rapid assay which is specific for a chosen enzyme which acts on a substrate to produce a single-stranded oligonucleotide product. This invention comprises a method of detecting the enzyme activity of a sample suspected of containing either an enzyme which acts on an oligonucleotide substrate to generate a single-stranded oligonucleotide product, or an inhibitor of such enzyme comprising: be assayed to generate a single-stranded oligonucleotide product; template, said DNA template comprising a first segment substantially complementary to the oligonucleotide product and a template 5'-extension region attached to the first segment, said extension region comprising at least one nucleotide, under hybridization conditions to form a RNA: DNA heteroduplex or a DNA:DNA duplex; segment of the DNA template; permitting the DNA polymerase to catalyze the addition of the labeled mononucleotide to the 3'-end of the oligonucleotide product to produce a labeled polymerase product; and amount of enzyme activity of the sample.
A further embodiment of this invention is a method of determining the site where an endonuclease cleaves an oligonucleotide substrate to create a cleavage product comprising: first 3'-segment putatively complementary to the cleavage product and of the same length as the cleavage product, and further comprising a 5'-extension region comprising at least one nucleotide; form a RNA:DNA heteroduplex or DNA:DNA duplex; the 5'-extension region; polymerase reaction to occur wherein the labeled nucleotide is added to the 3'-end of the cleavage product if the 3'-segment of the DNA template is complementary to the cleavage product and of the same length as the cleavage product; and
In accordance with this invention, the sample to be analyzed may contain an unknown quantity of an enzyme which acts on an oligonucleotide substrate (either DNA or RNA) to produce a single-stranded oligonucleotide product. Examples of such enzymes include various endonucleases, including either DNA or RNA endonucleases and ribozymes. Especially preferred are viral endonucleases and ribozymes.
In one embodiment of the invention, the amount of enzyme present in a sample may be quantified. In an alternative embodiment, the sample may contain a known amount of enzyme, and a substance whose inhibitory activit
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AMERSHAM Life Sciences Catalogue, 1993.
Cole James L.
Kuo Lawrence C.
Olsen David B.
Guzo David
Larson Thomas G.
Merck & Co. , Inc.
Tribble Jack L.
Yablonsky Michael D.
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