Detection of reverse transcriptase by DNA hybridization assay

Details Detection of reverse transcriptase by DNA hybridization assay Detection of reverse transcriptase by DNA hybridization assay

1992-07-14

2001-11-13

Myers, Carla J. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S091200, C435S091510, C435S091100, C435S015000

Reexamination Certificate

active

06316182

ABSTRACT:

FIELD OF THE INVENTION
This invention is related to the detection of reverse transcriptase and, more specifically, to the following: a) detection of such enzymes by allowing expression of their enzymatic activity followed by the detection of the resulting DNA; b) identification of drug-resistant reverse transcriptase mutants in samples; and/or c) screening of compounds for reverse transcriptase inhibitory activity.
BACKGROUND OF THE INVENTION
Retroviruses are enveloped viruses with a single stranded RNA genome in which the virion contains a reverse transcriptase (RT) enzyme essential for viral replication. All known reverse transcriptases are multifunctional with three different enzymatic activities: RNA-dependent DNA polymerase, RNase H, and DNA-dependent DNA polymerase. During retroviral replication, the first one of these, RNA-dependent DNA polymerase activity, generates a DNA (cDNA) strand complementary to the viral RNA. This first step is followed by the degradation of the original viral RNA strand by RNase H and then the DNA-dependent DNA polymerase activity helps generating a second DNA strand complementary to the first. This double stranded DNA is then integrated into the host genome leading to the ultimate replication of the virus.
Since the original discovery of reverse transcriptase in 1970, and in recognition of its important role, RT is frequently measured as an indication of the presence of a retrovirus. Detection of RT can be very important because this enzyme is a logical target for anti-viral therapy and, therefore, anti-viral drug screening can be carried out by RT detection.
One of the challenges facing clinicians in using anti-viral agents to treat patients is the development of mutant viruses resistant to those antivirals. The problem is especially acute in the development of anti-HIV agents. HIV is the causative agent of AIDS.
Thus, it is also important to clinicians to have the ability to determine whether RT in a clinical isolate is resistant and to what level to a particular drug or drugs.
RT can be detected directly as by the detection of certain viruses or indirectly by the detection of certain nucleic acids. One current method for detecting RT includes the use of a poly (rA) template and an oligo (dT) primer, approximately 15-20 nucleotides long, in presence of tritiated TTP (
3
H-TTP). After the formation of the template primer complex, primer extension is carried out in the presence of tritiated TTP followed by cold trichloroacetic acid precipitation of the DNA and RNA. Then, after a centrifugation or filtration and wash steps, scintillation counter measurement of the resuspended pellet, to determine the presence and the amount of incorporated
3
H-TTP, provides an indication of the presence of RT in the original sample. An alternative to this method is the use of
32
P-TTP, instead of
3
H-TTP. One such test is described by Krug et al., Meth. Enz., Vol. 152, 316 (1987).
Yet another alternative method for the measurement of RT is described in EP 0 392 459, published Oct. 17, 1990. This European patent application discloses a method for the determination of the presence of RT in which an artificially prepared adenine ribopolynucleotide RNA template and an oligodeoxythyminenucleotide primer are reacted with a biotinylated deoxyuridine triphosphate in the presence of a sample. After separation of the product from unreacted biotinylated reagent, the amount of incorporated biotin is measured.
The VII International Conference on AIDS, Abstract No. M.A. 1084, Jun. 16-21, 1991, describes an ELISA for measuring RT activity which involves using biotin and dioxigenin labeled nucleotides. The polymerization product is immobilized on a streptavidin-coated microtiter plate via the biotin/streptavidin interaction. RT activity is measured using a peroxidase-labeled anti-dioxigenin antibody which binds to dioxigenin-labeled cDNA.
Similarly, Konig et al., Journal of Cellular Biochemistry, Keystone Symposia on Molecular and Cellular Biology, Supplement 16E, Mar. 27-Apr. 4, 1992, Abstract No. Q222, describes a reverse transcriptase assay using biotin and dioxigenin as described above to screen compounds for activity as reverse transcriptase inhibitors.
These methods for RT detection are in general tedious, time consuming and, in most instances, generate hazardous waste. Additionally, such methods are not entirely specific because the non-reverse transcriptases may also generate a DNA duplex when the substrate (template) utilized is a homopolymer.
There exists a need for a convenient and rapid method for RT detection without generating hazardous wastes. There also exists a need to evaluate whether reverse transcriptase in a sample is resistant to any reverse transcriptase inhibitor(s) and to screen compounds for activity as reverse transcriptase inhibitors.
SUMMARY OF THE INVENTION
In one embodiment this invention concerns a method for detecting reverse transcriptase in a sample suspected of containing reverse transcriptase comprising the steps of:
(A) incubating a synthetic heteropolymeric RNA template molecule containing 40-500 nucleotides with an oligonucleotide primer complementary to a portion of said RNA molecule and of sufficient length to form a stable template-primer complex;
(B) contacting the complex formed in step (A) with a sample suspected of containing reverse transcriptase under conditions leading to the production of a cDNA strand complementary to the template RNA and hybridized thereto if reverse transcriptase was present;
(C) degrading the RNA template from the RNA-cDNA complex formed in step (B) resulting in single stranded cDNA;
(D) hybridizing said cDNA with a chemically modified oligonucleotide probe or probes to permit capture and/or detection of the cDNA-probe complex formed;
(E) separating the cDNA-probe complex formed in step (D) from unreacted probe, wherein steps (D) and (E) can be carried out sequentially or simultaneously; and
(F) detecting a label in a labeled cDNA complex, wherein said label is introduced into said complex from labeled oligonucleotide primer, labeled oligonucleotide probe or a labeled third oligonucleotide when the primer is unmodified.
In the methods described herein, the oligonucleotide primer can have one of the following three formats: first, it can be a labeled oligonucleotide, in which case the chemically modified oligonucleotide is an oligonucleotide to which is attached a capture hapten; second, an oligonucleotide with an attached capture hapten in which case the chemically modified oligonucleotide is the labeled oligonucleotide; and third, an unlabeled oligonucleotide in which case the assay contains both an oligonucleotide with an attached capture hapten and a labeled oligonucleotide.
In a second embodiment, this invention concerns a method for detecting and/or quantitating drug resistance of reverse transcriptase in a sample which comprises:
(A) incubating a synthetic heteropolymeric RNA template molecule containing 40-500 nucleotides with an oligonucleotide primer complementary to a portion of said RNA molecule and of sufficient length to form a stable template-primer complex;
(B) contacting the complex formed in step (A) with a sample containing reverse transcriptase and at least one drug known to have reverse transcriptase inhibitory activity under conditions leading to the production of a cDNA strand complementary to the template RNA and hybridized thereto if the reverse transcriptase was not inhibited by the drug;
(C) degrading the RNA template from the RNA-cDNA complex if formed in step (B) resulting in single stranded cDNA;
(D) hybridizing said cDNA if formed with a chemically modified oligonucleotide probe or probes to permit capture and/or detection of the cDNA-probe complex formed;
(E) separating the cDNA-probe complex if formed in step (D) from unreacted probe, wherein steps (D) and (E) can be carried out sequentially or simultaneously; and
(F) detecting and/or quantitating a label in a labeled cDNA complex, wherein said label is introduced into said complex from labeled oligonucleotide primer, l

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