Article pubs.acs.org/ac
Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time K. K. Sharma,† F. Przybilla,† T. Restle,‡ C. Boudier,† J. Godet,†,§ and Y. Mély*,† †
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie, 74 route du Rhin, 67401 Illkirch, France ‡ Institute für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Schleswig-Holstein, Germany § Département d’Information Médicale et de Biostatistiques, Hôpitaux Universitaires de Strasbourg, 1, pl de l’Hôpital, 67400 Strasbourg, France S Supporting Information *
ABSTRACT: Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radiolabeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT’s binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.
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Besides structural studies, a number of assays have been developed to monitor RT polymerization activity in vitro. Polymerase assays using 3H or P32-labeled reactants or modified nucleotide analogues were developed to monitor the products generated during the course of primer elongation/dNTP incorporation.9−12 In parallel, assays based on calorimetry 13−19 or Product Enhanced Reverse Transcriptase (PERT)20−24 were also developed. However, most of these assays, barring few,25,26 involved discontinuous multisteps which make them cumbersome, expensive, and difficult to scale up. On the other hand, continuous assays developed until now, except few,12 have relatively high background signal that limit their sensitivity.25,26 Nevertheless, these assays paved the way for the development of the numerous nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) that are on the
uman immunodeficiency virus type 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). During the HIV-1 replication cycle, the (+) strand viral RNA genome is reverse transcribed by the HIV-1-encoded reverse transcriptase (RT) to form an integration-competent double-stranded DNA.1,2 RT is a heterodimer consisting of a 66-kDa (p66) and a 51-kDa (p51) subunit, the latter being a truncated form of the former. The p66 sequence can be divided into thumb, palm, fingers, connection, and RNase H domains.3,4 Whereas p51 plays a structural role, p66 exhibits RNA-and DNA-dependent DNA polymerase activity5 as well as a hydrolytic (RNase H) activity. Due to its critical role in virus replication, RT has been extensively investigated. A large collection of RT structures have been reported including about 20 wild-type RT structures with nucleic acid substrates in binary or ternary complexes with deoxynucleotide triphosphate (dNTP), thus representing different steps of the DNA synthesis process (reviewed in refs 6−8). © XXXX American Chemical Society
Received: March 24, 2015 Accepted: June 30, 2015
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DOI: 10.1021/acs.analchem.5b01126 Anal. Chem. XXXX, XXX, XXX−XXX
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sulfoxide (DMSO). AZT triphosphate (3′-azido-3′-deoxythymidine-5′-triphosphate) was purchased from Genaxxon bioscience (Germany). Oligonucleotides. Synthetic purified oligodeoxynucleotides were purchased from IBA (Göttingen, Germany). Their concentrations were determined by UV absorbance at 260 nm. FRET measurements were carried out with a 17-mer (5′UUAAAAGAAAAGGGGGG) RNA-primer annealed to a 40mer (3′-AATTTTCTTTTCCCCCCTAACCCCCCATGTCACGTCCCCT) template. A carboxytetramethylrhodamine (TMR) dye was covalently attached via a C6 amino linker to the 3′ end of the template. Primer and template oligodeoxynucleotides were annealed by heating equimolar amounts at 90 °C for 2 min, followed by cooling to room temperature over several hours in a heating block. Unless noted otherwise, all experiments were carried out at 20 °C in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM KCl, and 6 mM MgCl2. Fluorescence Measurements. All steady-state and kinetic experiments were performed by using Alexa448-labeled RT mutant and TMR-labeled p/t duplexes. Formation of RT/ substrate complexes resulted in FRET from Alexa488 to TMR. All measurements were performed at 20 °C using either a spectrofluorometer (Fluorolog or Fluoromax-3 - Jobin Yvon) or a stopped-flow apparatus (Bio-Logic SFM3). Excitation wavelength for the labeled RT was 480 nm. For kinetic experiments, Alexa488 emission was recorded by using a bandpass filter at 525/39 nm. Nucleotide incorporation kinetics were triggered by addition of deoxyribonucleotides (dNTP) in excess to a preincubated mixture of RT and p/t duplexes at equimolar concentrations. When polymerase kinetics were recorded in the presence of RT-inhibitors, the inhibitor was preincubated with RT and p/t duplexes, unless otherwise stated. The kinetics were fast enough to monitor the fluorescence intensities continuously without photobleaching (Figure S1B). The apparent rate constants kobs and the relative contributions of the kinetic components were determined from the experimental progress curves, where the fluorescence intensity of Alexa488 was recorded as a function of the time. All fitting procedures were carried out with Origin 8.6 software using nonlinear least-squares methods and the Levenberg− Marquardt algorithm. Measurements in High Throughput Screening (HTS) Format. Experiments were performed in a 96-well plate. The complex of RT with p/t was formed by mixing 100 nM Alexa448-labeled RT mutant and 100 nM TMR-labeled p/t duplex in a total volume of 200 μL. All measurements were performed at 20 °C using a FLX-Xenius plate reader (Safas Monaco). Excitation wavelength was 480 nm, and emission was recorded at 520 nm by using 10 nm slits. Polymerization kinetics were triggered by addition of 100 μM dNTPs to the complex of RT with p/t. When the kinetics were recorded in the presence of RT-inhibitors, the inhibitor was preincubated with the complex of RT with p/t for at least 5 min, unless otherwise stated. Each set of experiments was performed in triplicate.
market. While both of these inhibitor classes can substantially slow down the progression of viral replication, over time these drugs lose their therapeutic efficacy due to the emergence of resistant strains of virus. Recently, single molecule experiments further highlighted the relationship between RT activity and its structural dynamics in RT-primer/template (p/t) complexes.27,28 RT was shown to dynamically flip between two orientations (polymerase conformation, when finger domain is toward 3′-OH of primer, or RNase H conformation, when RNase H domain is toward 3′-OH of primer)29,30 in order to perform either its polymerase or ribonuclease activity. RT flipping was mainly defined by the RNA or DNA nature of the template and the presence of cognate dNTP.29,30 These studies were performed by labeling the RT p66 subunit either at the RNase H domain or at the finger domain by a FRET donor and the nucleic acid substrate by a FRET acceptor. The binding orientation (polymerase vs RNase H) and dynamics of RT on these nucleic acid substrates were monitored through the time-dependent changes in the observed FRET values. Taken together, these studies highlighted the critical role of RT dynamics and binding orientation on its enzymatic activity. In this context, our objective was to determine whether the FRET-based strategy developed in single molecule experiments could be used in conventional ensemble fluorescence techniques to design a simple and real-time screening assay sensitive to the orientation and dynamics of RT on its substrate, as well as to RT polymerase activity. For this assay, we used RT labeled in its p51 thumb domain by Alexa 488, used as a FRET donor and a substrate comprising a 17-nucleotide RNA primer, annealed to a longer complementary DNA template labeled at its 3′ end by TMR, used as a FRET acceptor. This one-step and one-pot assay was found to be highly sensitive to RT orientation and polymerization, allowing an easy discrimination of NRTIs from NNRTIs and the determination of their half maximal inhibitory concentration (IC50) values. Consequently, the developed assay appears as a simple and robust alternative to the currently used assays for screening and validating antiRT drugs.
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MATERIALS AND METHODS Proteins. Mutant recombinant heterodimeric HIV-1BH10 RT was expressed in Escherichia coli and purified as described previously.31 The expression system and purification protocol allowed the preparation of large quantities of active heterodimeric enzyme. The RT mutant used in this study contains a single accessible cysteine at position 281 of the p51 thumb subdomain.32 This cysteine residue was labeled with Alexa488-C5 maleimide33 through a thiol-maleimide reaction34,35 with 87% labeling efficiency. Moreover, an E478Q mutation was introduced into the RNase H domain to abolish its RNA cleavage activity and, thus, prevent the RT-induced degradation of nucleic acid substrates.36 The polymerase activity of this mutant was comparable to that of the wildtype heterodimeric HIV-1BH10 RT (Figure S1A). Proteins were stored at −80 °C. Protein concentrations were determined using an extinction coefficient of 260, 450 M−1 cm−1 at 280 nm. Nucleotides and RT-Inhibitors. The deoxynucleotides (dNTP) mix was prepared by mixing each of UltraPure dATP, dCTP, dGTP and dTTP, purchased from Sigma-Aldrich (CAS number SLBF2196). Stock solutions of Nevirapine (11cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one) (Sigma-Aldrich) were prepared in dimethyl
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RESULTS AND DISCUSSION Assay Design. In order to build up an assay sensitive to both RT orientation and polymerase activity, we selected the central-Poly Purine Tract (cPPT) primer that together with its complementary DNA template is resistant to the nuclease activity of RT and mimics the synthesis steps of the pol gene during plus strand synthesis of HIV-1 genome.8,37,38 To be B
DOI: 10.1021/acs.analchem.5b01126 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Schematic representation of the designed FRET-based assay. The primer/template duplex consists of a 17-nucleotide cPPT primer annealed with a 40-nucleotide DNA template labeled with TMR (acting as FRET acceptor) at its 3′end. Due to the RNA nature of the primer, RT orients itself primarily in RNase H conformation, thus providing low FRET between TMR and Alexa488 (acting as FRET donor) covalently bound at K281C of the RT p51 domain. Addition of dNTP leads first to flipping of RT from RNase H orientation to polymerase orientation, inducing an increase in FRET. Then, by extending the primer, the RT polymerase activity increases the distance between the two fluorophores, so that FRET decreases.
where kobs1,2 are the observed kinetic rate constants, and I0 and IF are the fluorescence intensity before dNTP addition and the final intensity at the plateau, respectively, while Ii corresponds to the fluorescence of the intermediate state. In this equation, (I0 − Ii)e(−kobs1t) describes the declining part of the progress curve while IF −(IF − Ii)e(−kobs2t) describes the subsequent uprising part of the curve. The kinetic curves of E.RNA in the presence of dNTP could be well fitted using eq 1 with kobs1 = 0.11 (±0.01) s−1 and kobs2 = 0.0082 (±0.0003) s−1. To confirm our interpretation, the E.RNA was reacted with only dATP, so that extension of the primer is limited to a single nucleotide. We observed a single exponential declining progress curve (Figure 2, blue trace), characterized by a rate constant value (0.31 ± 0.02 s−1), comparable to the kobs1 value. As extension of the primer by a single nucleotide is expected to only marginally change the distance between the two probes, the observed increase in FRET (from 40% to 57%) is thought to be mainly related to RT flipping from its RNase H to its polymerase orientation. This observed change in fluorescence confirms that flipping of RT is coupled to the binding and incorporation of the first correct cognate nucleotide.29 This conclusion is further substantiated by the similarity in the rate constant value with the usually reported one (0.1−0.4 s−1) for single nucleotide incorporation by RT.42−44 Noticeably, as the RT molecules are not fully synchronized in our ensemble FRET assay, being in different orientations and positions along the elongated primer/ template, the observed FRET value at a given time point corresponds to an average of the FRET values from all RT molecules in solution. Moreover, as we did not observe any increase in donor fluorescence after reaching plateau, RT likely
sensitive to both changes in RT orientation on its substrate and extension of the primer, we used a 40-nt DNA template labeled at its 3′ end by TMR annealed to a 17nt RNA primer (Figure 1). RT was labeled by Alexa488 at position K281C in the p51 subunit, close to the RNase H domain of RT. As RT binds in its RNase H conformation to RNA/DNA substrates, a large distance between Alexa488 and TMR, and thus a low FRET, is expected as a result of the initial binding. As dNTP could not be incorporated while RT remains in RNase H orientation, addition of dNTP should first lead to flipping of RT from RNase H orientation to polymerase orientation, resulting in a closer proximity of the two dyes and, thus, higher FRET. Finally, as a result of the primer extension by RT polymerase activity, a progressive increase of the distance between the two probes and, thus, a progressive FRET decrease is expected. Addition of the TMR-labeled p/t to the Alexa488-labeled RT induces a 40% FRET (Figure 2, inset), indicating the formation of a complex between RT and p/t (hereinafter referred to as E.RNA). Next, as expected, addition of dNTP to E.RNA was observed to induce a decrease in donor fluorescence and, thus, an increase in FRET to 51%, during the first 10 s−30 s, likely as a result of RT flipping on its substrate (Figure 2, red trace). Then, in further line with our expectations, we observed a progressive increase in donor fluorescence, reaching a plateau at 25% FRET in about 200 s, after completion of the primer extension by RT. The obtained progression curves are typical of a difference of two exponential functions39−41 I(t ) = (I0 − Ii)e(−kobs1t ) − (IF − Ii)e(−kobs2t ) + IF
(1) C
DOI: 10.1021/acs.analchem.5b01126 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
green trace), showing the same rate constant (0.011 (±0.001) s−1) and the same plateau than the uprising part of E.RNA in the presence of all dNTP (Figure 2, red trace). As expected, the observed rate constant is similar to the primer extension rate constant of the DNA/RNA hybrid duplex, determined by single-molecule assay and gel electrophoresis.30 Next, we explored the dependence of the kobs values on the dNTP concentrations to further identify the mechanistic steps monitored by this assay (Figure S2). Through this dependence, our assay was suggested to monitor the formation of the final extended duplex via two parallel pathways that depend on the initial orientation of RT. Taken together, our data indicate that the designed assay allows following RT orientation and polymerase activity, using conventional ensemble measurements. The kobs1 rate constant of this assay mainly depends on the flipping-coupled incorporation of the first dNTP, while the kobs2 rate constant describes the primer extension. Therefore, this assay could be easily used to screen RT inhibitors, interfering with one of the two steps or both steps. Noticeably, it was reported that RT polymerase activity could be also monitored using another fluorescence-based assay using the RT-induced fluorescence enhancement resulting from the nonspecific interaction of nonlabeled RT with a Cy3 probe on the 5′-terminus of the primer.51 In this assay, the distance-dependence of the fluorescence drop as a result of primer extension is much sharper than in our FRET-based assay, so that only incorporation of a few nucleotides (
