WO1996024694A1

WO1996024694A1 - Method and kit for fluorometric analysis of enzymes catalyzing synthesis of nucleic acids

Info

Publication number: WO1996024694A1
Application number: PCT/US1996/001836
Authority: WO
Inventors: Surendra J. Chavan; Hans J. Prochaska
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 1995-02-10
Filing date: 1996-02-09
Publication date: 1996-08-15
Also published as: AU5022596A

Abstract

This invention provides a method for detecting in a sample an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA. Also provided is a method for determining in a sample the activity of such an enzyme. This invention also provides a method for detecting an RNA-DNA heteroduplex in a sample which comprises contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA. This method for detecting an RNA-DNA heteroduplex may further comprise quantitatively determining detected RNA-DNA heteroduplex. This invention also provides a method for detecting in a sample en enzyme which catalyzes the synthesis of and RNA-DNA heteroduplex from a nucleic acid template, as well as a method of detecting the activity of such an enzyme in a sample. This invention further provides a method for detecting the viral load of HIV in a sample. Also provided is a method for diagnosing an HIV infection in a subject, and for monitoring the progression of an HIV infection in a subject. Also provided is a method for determining the viral load of HIV in a subject infected with HIV. This invention further provides a method for identifying whether a substance inhibits reverse transcriptase. Finally, this invention provides a kit for assaying an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template.

Description

METHOD AND KIT FOR FLUO OMET IC ANALYSIS OF ENZYMES CATALYZING SYNTHESIS OF NUCLEIC ACIDS

This application is a continuation of U.S. Serial No. 08/386,469, filed February 10, 1995, the contents of which are hereby incorporated by reference into the present application.

Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of this application, preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background of the Invention

The epidemic of human immunodeficiency virus (HIV) infection and AIDS continues to grow in the United States and world-wide (25) . Although an enormous body of basic information concerning viral pathogenesis and life cycle has been described (26,27) , mortality due to AIDS continues to rise.

With better understanding of the HIV life cycle and HIV-host cell interactions have come strategies for interrupting viral replication (28) . Theoretically, any step in the HIV life cycle is susceptible to pharmacologic attack and many targets for antiviral intervention have been investigated (29) . The virus- encoded reverse transcriptase (RT) remains the most accessible of the targets under investigation (23) . To date, four nucleoside analog inhibitors of reverse transcription have received approval for marketing in the United States, with other nucleoside and non-nucleoside compounds in advanced clinical trials. The search for potent inhibitors of RT continues to be a major focus of research. In addition, although p24 antigen levels in culture supernatants is routinely used as a surrogate marker for viral replication in in vitro culture systems for screening inhibitors of viral replication, RT activity in culture supernatants has and continues to be used as a surrogate marker of viral load (30,7) . We have developed a rapid, efficient, and inexpensive assay fo measuring RT activity using a novel fluorometric approach. All antiretrovirals currently approved for use in the United States for the treatment of human immunodeficiency virus type 1 (HIV-1) infection are inhibitors of RT (1) , and the search for potent inhibitors of this enzyme continues to be a major focus of research. Although conventional isotopic methods for RT activity are highly sensitive, they all suffer from the following significant disadvantages which limit their utility: (a) the performance of these assays is limited to laboratories that hold appropriate licenses for radioactivity; (b) they are highly labor-intensive and time-consuming procedures; (c) the disposal of large quantities of scintillation fluid and radioactive isotopes required for these assays is a growing environmental concern; and (d) there are many sources of potential errors since the assays are complex (these procedures involve steps wherein the RNA-DNA heteroduplex produced by RT catalysis is precipitated or bound to filters and washed free of the radiolabeled substrate) . Nonradiometric assays that measure incorporation of nucleotides by RT into DNA by immunological methods (2-5) do not suffer these disadvantages. Unfortunately, commercially available kits based on these immunological detection methods are very costly (-$400 per 96-well plate) .

Summary of the Invention

This invention provides a method for detecting in a sample an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalysis by the enzyme; (b) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to double-stranded nucleic acid molecules in the sample synthesized by the catalysis; and (c) detecting the presence of fluorescence in the sample resulting from step (b) , thereby detecting in the sample the enzyme.

This invention also provides a method for determining in a sample the activity of an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalysis by the enzyme; (b) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to double-stranded nucleic acid molecules in the sample synthesized by the catalysis,- (c) quantitatively measuring fluorescence in the sample resulting from step (b) ; and (d) calculating the activity of any enzyme in the sample as a predetermined function of the quantity of fluorescence measured in step (c) .

The subject invention also provides a method for detecting an RNA-DNA heteroduplex in a sample which comprises: (a) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to the RNA-DNA heteroduplex in the sample; and (b) detecting the presence of fluorescence in the sample resulting from step (a) .

The subject invention also provides the aforementioned method of detecting an RNA-DNA heteroduplex in a sample, which method further comprises quantitatively measuring fluorescence detected in step (b) so as to quantitatively determine the RNA-DNA heteroduplex in the sample.

The subject invention further provides a method for detecting in a sample an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalyses of RNA-DNA heteroduplexes by the enzyme; and (b) detecting the presence of RNA-DNA heteroduplexes in the sample resulting from step (a) according to the aforementioned method of detecting an RNA-DNA heteroduplex in a sample, thereby detecting in the sample the enzyme.

The subject invention further provides a method for determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalyses of RNA-DNA heteroduplexes by the enzyme; (b) quantitatively determining RNA-DNA heteroduplexes in the sample resulting from step (a) according to the aforementioned method of quantitatively determining an RNA-DNA heteroduplex in a sample; and (c) calculating the activity of any enzyme in the sample as a predetermined function of the quantity of RNA-DNA heteroduplex determined in step (b) .

This invention also provides a method for determining in a sample viral load of HIV which comprises: (a) determining the activity of reverse transcriptase in the sample according to the aforementioned method of determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template; and (b) calculating the viral load of HIV in the sample as a predetermined function of the activity of reverse transcriptase determined in step (a) .

The subject invention also provides a method for diagnosing an HIV infection in a subject which comprises: (a) obtaining a suitable sample from the subject; and (b) detecting the presence of reverse transcriptase in the sample according to the aforementioned method for detecting in a sample an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template, the presence of reverse transcriptase indicating an HIV infection.

The subject invention further provides a method for determining the viral load of HIV in a subject infected with HIV which comprises: (a) obtaining a suitable sample from the subject; and (b) determining the viral load of HIV in the sample according to the aforementioned method for determining in a sample viral load of HIV, thereby determining the viral load in the subject.

The subject invention further provides a method for monitoring over a period of time the progression of an HIV infection in a subject infected with HIV which comprises: (a) determining the viral load of HIV in the subject according to the aforementioned method for determining the viral load of HIV in a subject at a plurality of points suitably spaced over the period of time, thereby determining a plurality of viral loads; and (b) comparing the viral loads determined in step (a) , thereby monitoring the progression of the HIV infection in the subject over the period of time.

The subject invention also provides a method for identifying whether a substance inhibits reverse transcriptase which comprises: (a) obtaining a sample comprising reverse transcriptase, the activity of the reverse transcriptase in the sample being predetermined; (b) contacting the sample with the substance; (c) determining the activity of reverse transcriptase in the sample resulting from step (b) according to the aforementioned method for determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template; and (d) ascertaining whether the activity determined in step (c) is less than the predetermined activity of reverse transcriptase in the sample obtained in step (a) , a lower activity in step (c) indicating inhibition of reverse transcriptase by the substance.

Finally, this invention provides a kit for assaying an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises: (a) substrates for initiating catalysis by the enzyme; and (b) a suitable fluorophore capable of selectively binding to double- stranded DNA.

Brief Description of the Drawings

Figure 1. Dependence of DAPI fluorescence on the amount of

HIV-1 RT. Purified recombinant HIV-1 RT was added in the indicated quantities to the RT assay described in the "Experimental Details" Section and incubated from 0 to 60 minutes at 37°C before stopping the reaction with EDTA. Aliquots were dispensed into cuvettes containing DAPI, and the resulting fluorescence was measured after 30 minutes incubation. The rate of fluorescence increase was linearly dependent on the amount of HIV-1 RT added to the RT assay up to 80 ng (r^s = 0.9997; inset) .

Figure 2 Comparison of the fluoro etric RT assay with an isotopic assay for RT activity. The fluorometric and isotopic assays were performed as described in the "Experimental Details" Section at the same time with the same preparation of purified recombinant HIV-1 RT. The concentration of the stock RT from which the dilutions was prepared was 0.5 mg/ml. The specific activity of RT from the experiment shown is calculated to be 630 ± 56 nmol dTTP incorporated/min/mg protein, and is consistent with the specific activity of our purified RT preparations. Both assays are highly correlated to each other over the same range of dilutions (1:250-1:5000) of RT (r² = 0.986) .

Figure 3. Stability of the fluorometric RT assay as a function of time. Pooled active fractions from the purification of RT (Table 1) were diluted 1:1000-1:5000 and were assayed in duplicate for RT activity by the fluorometric RT assay. In addition to assessing the fluorescence at 30 minutes incubation with DAPI, the cuvettes were kept in the dark and the fluorescence was reassessed at the time points indicated. The fluorescence at each time point was normalized to the fluorescence determined at 30 minutes, and the mean ± SE percentage fluorescence, represented by the symbols, was determined.

Figure 4, Effect of AZT 5' -triphosphate, nevirapine, and oltipraz on HIV-l RT activity as measured by DAPI fluorescence. Assays were carried out as described in the "Experimental Details" Section. Top, plot of 1/velocity as a function of AZT 5'- triphosphate appears to indicate competitive inhibition of HIV-l RT. This is confirmed by the negligible y-intercept of the corresponding Segel plot (inset; r² = 0.999) . The K_x was determined to be 13.8 nM for the experiment shown. Middle, plot of 1/velocity as a function of nevirapine concentration appears to show noncompetitive inhibition with respect to dTTP. However, the Segel transformation (inset; r² = 0.997) of these data indicates that the inhibition is mixed (α=0.76) , and the K was determined to be 25.8 μM. The two other experiments with nevirapine also showed mixed inhibition. Bottom, time- and concentration-dependent inhibition of HIV-l RT by oltipraz. The Kitz-Wilson transformation is shown in the inset (r² = 0.874) . The K, and k₃ for this experiment was determined to be 28.5 μM and 0.074 h^"1, respectively.

Detailed Description of the Invention

In the subject method, the fluorophore detects an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template by binding to the product of the catalysis, i.e. by binding to the double-stranded nucleic acid molecule synthesized by the addition of nucleotides or nucloesides to the nucleic acid template. The nucleic acid template may be any single-stranded nucleic acid molecule, such as single-stranded RNA or DNA. The nucleic acid template may be a naturally-occurring nucleic acid, for example DNA found in a cell, or it may be a synthetic nucleic acid, such as synthetic poly-dT or poly-A. The double-stranded nucleic acid may thus be a double-stranded DNA molecule, a double-stranded RNA molecule, or an RNA-DNA heteroduplex.

Enzymes which catalyze the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template are well-known to those of ordinary skill in the art, and any such enzyme may be detected by the subject method. Examples of enzymes which catalyze the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template include, but are not limited to, DNA polymerases, such as reverse transcriptase (RT) of DNA Polymerase I, which catalyze the synthesis of a DNA molecule from a DNA template; and RNA polymerase, which catalyzes the synthesis of an RNA molecule from a DNA template.

Any fluorophore which is capable of binding to double-stranded DNA may be used in the subject invention. In a double-stranded DNA molecule, such fluorophores bind to the minor groove of the molecule. Positively charged regions on the fluorophore are thought to bind to the negatively charged phosphate backbone in the minor groove. Binding to the minor groove prevents the transfer of a proton to the fluorophore which occurs in solution and quenches fluorescence. Prevention of this proton transfer causes these fluorophores to fluoresce. Without limiting the subject invention, it is believed that such fluorophores also bind to a minor groove in RNA-DNA heteroduplexes, resulting in fluorescence. Fluorophores which bind to double-stranded DNA molecules are well known in the art and include, but are not limited to, 4' ,6-diamidino-2-phenylindole; acridine orange; acridine homodimer; acridine-ethidium heterodimer; 9-amino-6- chloro-2-methoxyacridine; aminoactinomycin-D; benzothiazolium-4- quinolinium dimer dyes; bisbenzamide dyes; and ethidium homodimer.

The fluorophore used in the subject method must be suitable for binding to the particular double-stranded nucleic acid molecule synthesized by the catalysis. Determination of suitable fluorophores for binding to a particular nucleic acid molecule is well known to those of ordinary skill in the art. The suitability of a fluorophore for binding to a particular double- stranded nucleic acid molecule depends on such known factors as the base composition of the double-stranded nucleic acid molecule. The composition of the double-stranded nucleic acid molecule in turn depends of the composition of the nucleic acid template used as a substrate for initiation of catalysis. For example, 4' ,6-diamidino-2-phenylindole (DAPI) preferentially binds to contiguous dA-dT base pairs. Accordingly, DAPI is particularly suitable for use in a method as described above in which a nucleic acid template comprised of contiguous A-T base pairs is used as a substrate for initiation of catalysis of double-stranded nucleic acid. Certain fluorophores are, however, known to be less preferential in their binding and are known to bind a double-stranded DNA molecule regardless of the molecule's composition. - 11 -

In the method of the subject invention, fluorescence may be detected using any known method. For example, fluorescence may be detected by visually assessing the sample for the presence of fluorescent light. Preferably, however, fluorescence is detected by means of a fluorometer.

For purposes of the subject invention, a sample is any sample in which the detection of an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template is desired. In one embodiment, the sample is a biological fluid. Biological fluids are well-known to those of ordinary skill in the art and include, but are not limited to, urine, semen, saliva, and fluid blood fractions such as serum or plasma. Biological fluids also include tissue culture supernatants, such as the supernatant from cultured peripheral blood mononuclear cells or from cell lines. In another embodiment, the sample is a tissue. Examples of tissue samples which may be used in the method of the subject invention include samples lymphoid organs, such as lymph nodes or spleen; and tissue fractions of blood, such as lymphocytes. For purposes of the subject invention, the sample may already be predetermined to comprise the enzyme, such as a sample of the purified enzyme. Conversely, it may be unknown whether or not the sample contains the enzyme.

In one embodiment of the method of the subject invention, the sample is obtained from a mammal. When the sample is obtained from a mammal, in a further embodiment the sample is obtained from a human.

Any conditions sufficient to initiate catalysis by the enzyme may be used to treat the sample in step (a) of the subject method. Such conditions are well-known to those of ordinary skill in the art and will depend on the particular enzyme whose detection is desired. For example, treating the sample in step (a) may comprise contacting the sample with substances such as substrates and cofactors required by the enzyme to catalyze synthesis of the product double-strand nucleic acid molecule. In the case of DNA polymerases, such as RT, such substances include a nucleic acid template, a primer molecule, deoxynucleoside triphosphates, magnesium, and manganese salts. In the case of RNA polymerase, such substances include, a nucleic acid template, ribonucleotide triphosphates, and magnesium.

In one embodiment of the subject method, the method furthe comprises treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to contacting the sample with the fluorophore. Treatments useful for termination of catalysis are well-known to those of ordinary skill and may depend on the particular enzyme whose detection is desired. Such treatments include, but are not limited to, contacting the sample with EDTA, contacting the sample with an inhibitor of the enzyme, or diluting the sample to a concentration at which reaction between the substrates and the nucleic acid template cannot occur.

This invention also provides a method for determining in a sample the activity of an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalysis by the enzyme; (b) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to double-stranded nucleic acid molecules in the sample synthesized by the catalysis; (c) quantitatively measuring fluorescence in the sample resulting from step (b) ; and (d) calculating the activity of any enzyme in the sample as a predetermined function of the quantity of fluorescence measured in step (c) .

As used herein, the activity of an enzyme in a sample is the number of reactions catalyzed in the sample per unit time. Enzymatic activity in a sample depends on factors including, but not limited to, the number of enzyme molecules present in the sample. As is known in the art, the number of reactions catalyzed per unit time may be evaluated by analyzing a variety of parameters. In the method of the subject invention, the number of reactions catalyzed per unit time is evaluated by analyzing the quantity of product, i.e. double-stranded nucleic acid, produced per unit time.

Accordingly, in the subject method, the activity of the enzyme is determined as a function of the quantity of fluorescence measured in step (c) , since the quantity of fluorescence indicates the quantity of fluorescing fluorophore molecules bound to double-stranded nucleic acid molecules in the sample. The functional relationship between the activity of the enzyme and the quantity of fluorescence may be arbitrary. For example, the enzymatic activity may be arbitrarily deemed to be equivalent to the quantity of the fluorescence. Alternatively, a quantity of fluorescence may be converted to enzymatic activity in terms of, for example, moles substrate consumed by generating a calibration curve of fluorescence with an enzyme preparation of known activity. As is known in the art, the fluorometer can be calibrated thereafter with the use of a stable fluorescent solution such as quinine sulfate.

As used herein, the quantity of fluorescence in a sample means the intensity of fluorescent light emitted by the sample. In the subject invention, the quantity of fluorescence may be quantitatively measured using any known method. For example, the fluorescence may be quantitatively measured visually by assessing the intensity of any fluorescence in the sample, the more intense the fluorescence, the greater the quantity of fluorophore bound to double-stranded nucleic acid molecules. Preferably, however, the fluorescence is quantitatively measured using a fluorometer.

In one embodiment of the subject method, the method further comprises treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to contacting the sample with the fluorophore. Treatments sufficient to terminate catalysis of an enzyme in a sample are well-known to those of ordinary skill in the art and are described above.

The subject invention also provides a method for detecting an RNA-DNA heteroduplex in a sample which comprises: (a) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to any RNA-DNA heteroduplex in the sample; and (b) detecting the presence of fluorescence in the sample resulting from step (a) .

Any fluorophore capable of selectively binding to double-stranded DNA may be used in the subject method for detecting RNA-DNA heteroduplexes present in a sample, and such fluorophores are well-known to those of ordinary skill in the art. In different embodiments, the fluorophore in the subject method is 4', 6- diamidino-2-phenylindole; acridine orange; acridine homodimer; acridine-ethidium heterodimer; 9-amino-6-chloro-2 - methoxyacridine; aminoactinomycin-D; a benzothiazolium-4- quinolinium dimer dye; a bisbenzamide dye; and ethidium homodimer.

The subject method may be used to detect RNA-DNA heteroduplexes in any sample in which detection of such heteroduplexes is desired. Samples in which RNA-DNA heteroduplexes may be detected according to the subject method include, but are not limited to, the examples of samples described above.

Detection of RNA-DNA heteroduplexes in a sample is accomplished by detecting the fluorophore bound to RNA-DNA heteroduplexes in the sample. As indicated above, binding of the fluorophore causes the fluorophore to emit fluorescent light. The fluorescence may be detected using any known method, including, but not limited to, visual detection or detection with a fluorometer.

The quantity of fluorescence depends on the quantity of fluorescing fluorophore molecules, which in turn depends on the quantity of RNA-DNA heteroduplex in the sample capable of binding the fluorophore. As used herein, the quantity of RNA-DNA heteroduplex in a sample is a function of both the number of RNA- DNA heteroduplex molecules and the length of each molecule, i.e. it corresponds to the quantity of conjugated base pairs in the sample. Accordingly, the subject invention also provides the above-described method of detecting RNA-DNA heteroduplexes present in a sample, which method further comprises quantitatively measuring fluorescence detected in step (b) so as to quantitatively determine the RNA-DNA heteroduplex in the sample. Methods for quantitatively measuring fluorescence are well known in the art, and any such method may be used in the subject invention. Such methods are described above.

The subject invention further provides a method for detecting in a sample an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalyses of RNA-DNA heteroduplexes by the enzyme; and (b) detecting the presence of RNA-DNA heteroduplexes in the sample resulting from step (a) according to the above-described method of detecting RNA-DNA heteroduplexes present in a sample, thereby detecting in the sample the enzyme.

In one embodiment, the method further comprises treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to detecting the presence of RNA-DNA heteroduplexes in the sample. Such treatments are well-known to those of ordinary skill in the art and are described above.

Any enzyme capable of catalyzing the synthesis of RNA-DNA heteroduplexes from a nucleic acid template may be detected according to the subject method. In one embodiment, the enzyme catalyzes the synthesis of an RNA-DNA heteroduplex from an RNA template, as does RT. In a different embodiment, the enzyme catalyzes the synthesis of an RNA-DNA heteroduplex from a DNA template, as in the case of RNA polymerase.

The subject invention further provides a method for determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template which comprises: (a) treating the sample under conditions sufficient to initiate catalyses of the RNA-DNA heteroduplex by the enzyme; (b) quantitatively determining the RNA-DNA heteroduplex in the sample resulting from step (a) according to the above-described method of quantitatively determining RNA-DNA heteroduplexes present in a sample; and (c) calculating the activity of any enzyme in the sample as a predetermined function of the quantity of the RNA-DNA heteroduplex determined in step (b) .

As indicated above, enzymatic activity in a sample means the number of reactions catalyzed in the sample per unit time. The number of reactions catalyzed is determinable by analyzing a variety of different parameters, for example the quantity of substrates consumed per unit time or the quantity of product produced per unit time. In the subject method, the number of reactions is determined by analyzing the quantity of product, i.e. the quantity of the RNA-DNA heteroduplex, produced. Measurement is accomplished by the determination of the quantity of fluorophore bound to RNA-DNA heteroduplex in the sample via fluorescence. Any method for measuring the quantity of fluorescence may be used, and such methods are described above.

In one embodiment, the method further comprises treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to quantitatively determining RNA-DNA heteroduplexes in the sample. Treatments sufficient to terminate catalysis by such enzymes are known in the art as described above, and any such treatment be used in the subject method.

In one embodiment, the enzyme whose activity is determined is reverse transcriptase. In another embodiment, the enzyme is RNA polymerase.

This invention also provides a method for determining in a sample viral load of HIV which comprises: (a) determining the activity of reverse transcriptase in the sample according to the above- described method of determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template; and (b) calculating the viral load of HIV in the sample as a predetermined function of the activity of reverse transcriptase determined in step (a) .

As used herein, the term viral load means virion concentration. In the subject method for determining viral load, the viral load may be calculated as an arbitrary function of the activity of reverse transcriptase determined in step (a) . For example, the viral load may arbitrarily, for purposes of comparing two or more different samples, be deemed to be equivalent to the activity of reverse transcriptase. Alternatively, the viral load may be calculated from the actual quantity of reverse transcriptase molecules calculated in the sample, since each HIV virion contains two reverse transcriptase molecules. As described above, the actual quantity of reverse transcriptase molecules in a sample may be determined by comparing the fluorescence resulting in the sample to the fluorescence produced by a standard predetermined t.o contain a specific quantity of reverse transcriptase molecules.

The subject invention also provides a method for diagnosing an HIV infection in a subject which comprises: (a) obtaining a suitable sample from the subject; and (b) detecting the presence of reverse transcriptase in the sample according to the above- described method for detecting in a sample an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template, the presence of reverse transcriptase indicating an HIV infection.

For purposes of the subject invention, a suitable sample is any sample obtained from a subject in which HIV virions would be present if the subject were infected with HIV. Such samples are well known in the art and include, but are not limited to, blood fractions, such as plasma, serum, or lymphocytes.

In one embodiment, the subject is a mammal. When the subject is a mammal, the subject may be a human being.

As indicated above, a suitable sample is any sample obtained from a subject in which HIV virions would be present if the subject were infected with HIV. Suitable samples which may be used in the method of the subject invention for determining viral load of HIV in a subject are described above.

The subject invention further provides a method for monitoring over a period of time the progression of an HIV infection in a subject infected with HIV which comprises: (a) determining the viral load of HIV in the subject according to the above-described method at a plurality of points suitably spaced over the period of time, thereby determining a plurality of viral loads; and (b) comparing the viral loads determined in step (a) , thereby monitoring the progression of the HIV infection in the subject over the period of time.

The subject invention also provides a method for identifying whether a substance inhibits reverse transcriptase which comprises: (a) obtaining a sample comprising reverse transcriptase, the activity of the reverse transcriptase in the sample being predetermined; (b) contacting the sample with the substance; (c) determining the activity of reverse transcriptase in the sample resulting from step (b) according to the above- described method for determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template; and (d) ascertaining whether the activity determined in step (c) is less than the predetermined activity of reverse transcriptase in the sample obtained in step

(a) , a lower activity in step (c) indicating inhibition of reverse transcriptase by the substance.

Samples comprising reverse transcriptase are well known and readily obtainable by those of ordinary skill. Samples containing reverse transcriptase may be commercially obtained. Also, techniques for obtaining a sample of reverse transcriptase are well known and include, but are not limited to, purifying the lysates of transformed bacteria containing expression plasmids for reverse transcriptase.

The activity of reverse transcriptase in the sample obtained in step (a) may be predetermined by any known technique for determining reverse transcriptase activity. The reverse transcriptase activity of some commercially-obtainable samples is provided by the manufacturer. In one embodiment of the subject method, the activity of reverse transcriptase in the sample obtained in step (a) is predetermined according to the above-described method of the subject invention for determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template.

As used herein, assaying includes merely detecting an enzyme, as well as measuring the activity of an enzyme. The subject kit is useful in any of the methods described herein. For example, the subject kit is useful for detecting an HIV infection in a subject, for determining the viral load of HIV in a subject infected with HIV, and for identifying whether a substance inhibits reverse transcriptase.

This invention will be better understood from the Examples in the "Experimental Details" Section which follows. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of, and are not intended to, nor should they be construed to, limit the invention as described more fully in the claims which follow thereafter.

Experimental Details Materials and Methods

Ma terials

DAPI (4' ,6-diamidino-2-phenylindole) was purchased fromMolecular Proves (Eugene, OR) ; Klenow fragment was obtained from Boerhinger-Mannheim (Indianapolis, IN); poly(A), oligo(dT) (n=10) , and poly (dA) -oligo(dT) (n=10) were purchased from Pharmacia (Picscataway, NJ) ; [α-³²P]dTTP (3000 Ci/m ol) was purchased from NEN (Boston, MA); AZT (3' -azido-3' -deoxythymidine) 5' -triphosphate was purchased form Moravek Biochemicals (Brea, CA) ; nevirapine [6,ll-dihydro-ll-cyclopropyl-4-methyldipyrido [2,3-b:2' ,3' -e] - [1,4]diazepine-6-one] was obtained fromDr. Bruce Polsky (Infectious Disease Service, Memorial Sloan-Kettering Cancer Center (MSKCC) ) . Oltipraz was synthesized as described (8) by Dr. William G. Bornmann (Preparative Synthesis CORE Facility, MSKCC) . All other chemicals and supplies were purchased from Fisher (Springfield, NJ) or Sigma (St. Louis, MO). Transformed bacteria containing expression plasmids for HIV-l and HIV-2 RT were generously provided by Dr. Amnon Hizi (Sackler School of Medicine, Tel Aviv, Israel) (9) . RT was purified to homogeneity utilizing a novel affinity purification approach (31) . Aliquots of pooled active fractions from the intermediate purification steps were stored at —70°C, whereas active fractions from the final step were concentrated and stored at —70°C in 1:1 glycerol: RT buffer [50 mM Tris-Cl (pH 7.8), 75 mM KC1, 5mM MgCl₂, 1 mM DTT, lrru EGTA, 0.1% Triton X-100, 0.1% NP-40, and 1 mg/ml bovine serum albumin (BSA) ] without BSA. Crude and pure enzyme preparations were diluted (1:100-1:5000) into RT buffer shortly before use.

Isotopic RT Assay

The method of Flexner et al. (10) was used with slight modification. RT buffer was dispensed (9 μl/well) and mixed with

(1 μl/well) diluted enzyme into a 96-well microtiter plate. The reaction was initiated with the addition of 25 μl/well RT buffer containing 10 μg/ml poly(A) , 2.5^' μg/ml oligo(dT), 5.6 μM dTTP, and 20 μCi/ml [α-³ P] dTTP. After incubation for 30 min at 37°C, the plates were placed on ice and 20 μl aliquots were spotte onto DE-81 paper and air-dried. The paper was then washed thre times in 2 X SSC (0.6 M NaCl, 0.06 M sodium citrate, pH 7.0) an dried, and the retained radioactivity was measured b scintillation counting.

Fluorometric Assay for RT Activi ty ■

The fluorometric assay for RT activity was derived from method for the spectrofluorometric determination of DNA (11,12) . R diluted in RT buffer was dispensed into wells of a microtite plate (40 μl/well) , and nonenzymatic blanks were prepared by the addition of 40 μl/well of RT buffer without RT. The reaction was initiated by the addition of 100 μl/well of RT reaction buffer

(RT buffer containing 20 μg/ml poly(A), 2 μg/ml oligo (dT), and

0-44.8 μM dTTP) . After incubating the microtiter stopped by the addition of the 40 μl/well of 0.5 M EDTA (pH 7.8), and 150 μl aliquots of the resulting mixture were transferred to clear four- sided methacrylate cuvettes (1 X 1 X 4.5 cm) that contained 1 ml of 23 nM DAPI in 5 ΓΠM Tris-Cl, 8 mM NaCl (pH 7.8) . The cuvettes were gently vortexed and incubated at room temperature in the dark for 30 min. The fluorescence was measured with a Perkin- Elmer 650S fluorescence detector (λ_excltatl0n = 359 nm; λ_emιsslon = 460 nm) . Enzymatic activities were determined by subtracting the fluorescence of the nonenzymatic blanks from the fluorescence of the enzyme-containing cuvettes.

Fluorometric Assays wi th AZT 5 ' -Triphosphate or Nevirapine

In experiments with either AZT 5' -triphosphate or nevirapine, 10 μl of RT buffer was added to all wells of a 96-well plate. AZT 5' -triphosphate or nevirapine at concentrations of 1.12 μM or 1.12 mM, respectively, were added to the top row of microtiter wells in a volume of 10 μl . After thoroughly mixing the contents of the well, 10 μl/well was removed and mixed with the contents of the second row of wells. These twofold serial dilutions were performed with all but the last two rows of wells (no inhibitor control row and blank control row) . RT buffer without RT was then added to the last row of wells (30 μl/well) , and the rest of the wells received 30 μl RT diluted into RT buffer. The reaction was initiated by the addition of 100 μl/well RT reaction buffer and was stopped at 30 min. The remainder of the assay procedure was carried out as described above. For K_α determinations, data was plotted according to the method of Dixon

(13) , and secondary transformations were generated according to the method of Segel (14) , from which K_A values were determined.

The IC₅₀ results were determined from the x-intercept of the line calculated by the linear regression of data plotted according to the median-effect principle of Chou and Talalay (15) .

Fluorometric Assay wi th Ol tipraz as an Inactivator of RT

RT was preincubated with various concentrations of oltipraz as described (6) . After incubation, 40 μl/well was transferred to a second microtiter plate and the remaining RT activity was assayed as described above. Data were analyzed according to the method described by Kitz and Wilson (16) .

Assay of DNA Polymerase Activi ty of Klenow Fragment

The isotopic and fluorometric assay of DNA polymerase activity of Klenow fragment was assayed as described above for RT, except that the poly(A) and oligo(dT) were substituted with 20 μl/ml of poly(dA) -oligo(dT) in the RT reaction buffer.

Results and Discussion

DAPI becomes highly fluorescent upon binding to the minor groove of B-DNA (17,18) and is a useful reagent for quantitating the DNA content of biological specimens (11,12). The compound has preferential binding affinity for contiguous dA-dT base pairs, and the increase in fluorescence intensity is thought to result from prevention of an intramolecular proton transfer in the excited state that occurs (and quenches fluorescence) in solution (18) . In preliminary experiments, we were able to demonstrate an increase in fluorescence after dTTP was incorporated into poly(A) -oligo(dT) by RT, which led us to evaluate whether the fluorescence of DAPI could be used to measure RT activity.

Figure 1 shows the increase in DAPI fluorescence as a function of incubation time at several concentrations of RT. As is apparent, the assay is linear with incubation time and the amount of RT present in the assay (Fig. 1, inset) . The time- and RT- dependent increase in fluorescence shown in Fig. 1 is completely abolished if: (a) RT is boiled for 5 min prior to dilution into RT buffer; (b) 40 μl/well of 0.5 M EDTA (pH 7.8) is added prior to the addition of RT reaction buffer; or (c) dTTP is omitted from the RT reaction buffer (data not shown) . This indicates that the increase in fluorescence is an accurate measure of RT activity. This was confirmed by simultaneously measuring the incorporation of [α-³P]dTTP into DNA and DAPI fluorescence with the same enzyme preparation. As can be seen in Fig. 2, the isotopic and fluorometric assays are exceedingly well-correlated to each other (r²=0.986).

We assessed whether RT activity could be detected in crude preparations of RT by asking whether the increase in the specific activity of RT during the course of the purification of the enzyme could be determined with the fluorometric assay (Table l) . Although an increase in DAPI fluorescence was easily detected in lysates from transformed bacteria expressing HIV-l RT (Table 1) , bacterial lysates that do not express RT had no effect upon DAPI fluorescence (data not shown) . The increase in specific activity of RT measures with the fluorometric assay correlated well with the increase in specific activity measured with the conventional isotopic assay (Table 1; r² = 0.988) . This is also reflected by the relatively constant ratio of the isotopic to the fluorometric assay for each of the purification steps (Table 1, last column) . Thus, the fluorometric assay can accurately measure RT activities from crude fractions, and we have found that the fluorometric assay can also detect the activity of RT during the chromatographic fractionation of the enzyme (data not shown) . Although we routinely measure the fluorescence after 30 min incubation with_.DAPI, we have determined that the increase in fluorescence from crude or purified fractions is stable for up to 24 h ( Fig . 3 ) .

The mean ± SE for the K^ of dTTP determined from Eadie-Hofstee plots with purified RT from four experiments was 7.71 ± 1.56 μM. This kinetic constant was easily determined with the fluorometric assay, was highly reproducible, and is within the range reported by other investigators (9, 19-21) . Moreover, the assay can be utilized to characterize the type and magnitude of RT inhibition. This is demonstrated in Fig. 4 with three known inhibitors of HIV-l RT. Although AZT 5' -triphosphate is a chain terminator of RNA directed DNA synthesis, its kinetic behavior is that of competitive inhibitor of RT with respect to dTTP. The K_± (11.1 ± 2.7 ΠM; N=2 experiments) determined with the fluorometric assay is similar to the K_A values published previously (22,23) . The K_t value for nevirapine was determined to be 13.1 ± 6.3 μ (N=3 experiments) , and is within the range of IC₅₀ or K_t results published previously utilizing standard radiometric assays with dTTP as substrate (19,21,24) . Although our Dixon plots appeared to indicate noncompetitive inhibition of RT with respect to dTTP (Fig. 4) , secondary Segel transformations were consistent with mixed-type inhibition (Fig. 4, middle frame, inset) . Mixed and/or noncompetitive inhibition has been observed with deoxynucleotide 5' -triphosphate substrates (24) . The ability of oltipraz to inactivate RT activity was also evaluated by the fluorometric assay. As expected (6) , addition of oltipraz to RT results in a time- and concentration-dependent loss of RT activity, which is kinetic evidence for the inactivation of the enzyme (16) . Both K and k₃ (25.0 ± 3.5 μM and 0.085 ± 0.011 h^"1, respectively; N=2 experiments) were similar to previously published results utilizing an isotopic assay for RT activity (6) .

We tested whether nevirapine and AZT 5' -triphosphate could inhibit the increased fluorescence of DAPI in assays conducted with HIV-2 RT. At 10 μM dTTP, the IC₅₀ for nevirapine was 2.94 ± 0.28 μM (N=4 experiments) when HIV-l RT was used as the enzyme source. As expected (20) , nevirapine had no effect on the fluorometric RT assay when HIV-2 RT was assayed under identical conditions (<3% inhibition at 160 μM nevirapine) . In stark contrast, the IC_S0 for AZT 5' -triphosphate in the presence of 10 μM dTTP with HIV-2 RT as the enzyme source was 74.2 ± 8.4 ΠM (N=3 experiments) . The IC₅₀ for AZT 5' -triphosphate under identical conditions with HIV-l RT was 15.2 ± 4.0 ΠM (N=3 experiments) . Thus, our fluorometric assay can distinguish inhibitors that are specific for HIV-l RT (e.g. nevirapine) .

Our invention provides a new and novel fluorometric assay for RT activity. Our goal was to develop a rapid assay for RT activity so that we could more readily study the inactivation of this enzyme by 1,2-dithiole-3-thiones. Although we have not systematically determined whether there are buffers or reagents that interfere with the assay, we have demonstrated that RT activity can be accurately detected from crude samples that differ widely in ionic strength and composition (Table 1) . We have determined that there is specificity for interaction between a polynucleotide duplex and a fluorescent probe with this assay. For example, we have established that the DNA polymerase activity of Klenow fragment cannot be detected by DAPI fluorescence when using poly(dA) -oligo(dT) as template -primer (data not shown; incorporation [α-³²P]dTTP into DNA was documented under our assay conditions) . Thus, the presence of 2'-hydroxyl groups in the poly(A) strand of poly(A) -poly(dT) allows for DAPI binding (and perturbation in DAPI fluorescence) . However, it is likely that another fluorescent probe could be found that would allow the fluorometric assessment of DNA polymerase activity with poly(dA) -oligo(dT) . By development of suitable fluorometric assays, screening compounds for their ability to inhibit RT or DNA polymerase activity will become far easier to perform. Moreover, the assay which we have described may be used to measure RT activity in biological samples for laboratory and/or clinical studies. TABLE 1

Comparison of RT Specific Activities Measured by the Conventional Isotopic RT Assay with the Fluorometric RT Assay

Isotopic Assay Fluorometric Assay

Puri fication RT spec act Fold RT spec act Fold a t i o o Step (nmol/min/mg) purification (F/mg) purification isotopic t f luorome ri

spec act

B a c t e r i a l lysate 2.8 =1 4.2 =1 0.67

( N H ₄ ) ₂ S 0 , fraction Before Dialysis 17 6.1 30 7.2 0.57

After Dialysis 21 7.4 43 10 0.49

Gel filtration 64 23 98 24 0.65

Dye - l igand chromatography 430 150 860 210 0.50

MonoQ ion exchange 630 230 1500 370 0.42

Note. Activities were measured on pooled active fractions from a purification procedure of RT (see footnote 1) fro transformed bacterial containing an expression plasmid for HIV-l RT (9) .

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Claims

What is claimed is:

1. A method for detecting in a sample an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises:

(a) treating the sample under conditions sufficient to initiate catalysis by the enzyme;

(b) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to double-stranded nucleic acid molecules in the sample synthesized by the catalysis; and

(c) detecting the presence of fluorescence in the sample resulting from step (b) , thereby detecting in the sample the enzyme .

2. The method of claim 1, further comprising treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to contacting the sample with the fluorophore.

3. A method for determining in a sample the activity of an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises:

(b) contacting the sample with a fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to double-stranded nucleic acid molecules in the sample synthesized by the catalysis;

(c) quantitatively measuring fluorescence in the sample resulting from step (b) ; and (d) calculating the activity of any enzyme in the sample as a predetermined function of the quantity of fluorescence measured in step (c) .

4. The method of claim 3, further comprising treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to contactin the sample with the fluorophore .

5. A method for detecting an RNA-DNA heteroduplex in a sample which comprises:

(a) contacting the sample with a suitable fluorophore which selectively binds to double-stranded DNA under conditions permitting said fluorophore to bind to the RNA-DNA heteroduplex present in the sample; and

(b) detecting the presence of fluorescence in the sample resulting from step (a) .

6. The method of claim 5, wherein the fluorophore is 4 ' ,6- diamidino-2-phenylindole; acridine orange; acridine homodimer; acridine-ethidium heterodimer; 9-amino-6-chloro- 2-methoxyacridine; aminoactinomycin-D; a benzothiazolium-4- quinoliniu dimer dye; a bisbenzamide dye; or ethidium homodimer.

7. The method of claim 6, wherein the fluorophore is 4 ',6- diamidino-2-phenylindole.

8. The method of claim 5, wherein the sample is a biological fluid.

9. The method of claim 5, wherein the sample is a tissue.

10. The method of claim 5, wherein the sample is obtained from a mammal .

11. The method of claim 10, wherein the mammal is a human.

12. The method of claim 5, further comprising quantitatively measuring fluorescence detected in step (b) so as to quantitatively determine the RNA-DNA heteroduplex in the sample.

13. A method for detecting in a sample an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template which comprises:

(a) treating the sample under conditions sufficient to initiate catalyses of RNA-DNA heteroduplexes by the enzyme; and

(b) detecting the presence of RNA-DNA heteroduplexes in the sample resulting from step (a) according to the method of claim 5, thereby detecting in the sample the enzyme.

14. The method of claim 13, further comprising treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to detecting the presence of RNA-DNA heteroduplexes in the sample.

15. The method of claim 13, wherein the enzyme is reverse transcriptase.

16. The method of claim 13, wherein the enzyme is RNA polymerase.

17. A method for determining in a sample the activity of an enzyme which catalyzes the synthesis of an RNA-DNA heteroduplex from a nucleic acid template which comprises:

(a) treating the sample under conditions sufficient to initiate catalyses of RNA-DNA heteroduplexes by the enzyme;

(b) quantitatively determining RNA-DNA heteroduplexes in the sample resulting from step (a) according to the method of claim 12; and

(c) calculating the activity of any enzyme in the sample as a predetermined function of the quantity of RNA-DNA heteroduplexes determined in step (b) .

18. The method of claim 17, further comprising treating the sample resulting from step (a) under conditions sufficient to terminate catalysis by the enzyme prior to quantitatively determining RNA-DNA heteroduplexes in th sample.

19. The method of claim 17, wherein the enzyme is reverse transcriptase.

20. The method of claim 17, wherein the enzyme is RN polymerase.

21. A method for determining in a sample viral load of HIV which comprises :

(a) determining the activity of reverse transcriptase in the sample according to the method of claim 19; and (b) calculating the viral load of HIV in the sample as a predetermined function of the activity of reverse transcriptase determined in step (a) .

22. A method for diagnosing an HIV infection in a subject which comprises:

(a) obtaining a suitable sample from the subject; and

(b) detecting the presence of reverse transcriptase in the sample according to the method of claim 15, the presence of reverse transcriptase indicating an HIV infection.

23. A method for determining the viral load of HIV in a subject infected with HIV which comprises :

(a) obtaining a suitable sample from the subject; and (b) determining the viral load of HIV in the sample according to the method of claim 21, thereby determining the viral load in the subject.

24. A method for monitoring over a period of time the progression of an HIV infection in a subject infected with

HIV which comprises :

(a) determining the viral load of HIV in the subject according to the method of claim 23 at a plurality of points suitably spaced over the period of time, thereby determining a plurality of viral loads; and (b) comparing the viral loads determined in step (a) , thereby monitoring the progression of the HIV infection in the subject over the period of time.

25. A method for identifying whether a substance inhibits reverse transcriptase which comprises:

(a) obtaining a sample comprising reverse transcriptase, the activity of the reverse transcriptase in the sample being predetermined;

(b) contacting the sample with the substance;

(c) determining the activity of reverse transcriptase in the sample resulting from step (b) according to the method of claim 19; and

(d) ascertaining whether the activity determined in step (c) is less than the predetermined activity of reverse transcriptase in the sample obtained in step (a) , a lower activity in step (c) indicating inhibition of reverse transcriptase by the substance.

26. The method of claim 25, wherein the activity of reverse transcriptase in the sample obtained in step (a) is predetermined according to the method of claim 19.

27. A kit for assaying an enzyme which catalyzes the synthesis of a double-stranded nucleic acid molecule from a nucleic acid template which comprises: (a) substrates for initiating catalysis by the enzyme; and (b) a suitable fluorophore capable of selectively binding to double-stranded DNA.