A Simple Colorimetric RNA Polymerase Assay

Virology 274, 429–437 (2000) doi:10.1006/viro.2000.0492, available online at http://www.idealibrary.com on

Exploiting Polymerase Promiscuity: A Simple Colorimetric RNA Polymerase Assay

William Vassiliou,* Jeffery B. Epp,† Bin-Bin Wang,* Alfred M. Del Vecchio,‡,1 Theodore Widlanski,§ and C. Cheng Kao*,2

*Department of Biology, Indiana University, Bloomington, Indiana 47405; †Dow AgroSciences, Indianapolis, Indiana 46268; ‡SmithKline Beecham Inc., Collegeville, Pennsylvania 19426; and §Department of Chemistry, Indiana University, Bloomington, Indiana 47405 Received May 18, 2000; accepted June 29, 2000

We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the ␥-phosphate were synthesized (PNP–NTPs). These PNP–NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP–NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis. © 2000 Academic Press

INTRODUCTION polymerases are primarily tested using radioactive assays that require special handling of the isotope RNA and DNA polymerases carry out the synthesis of and are incompatible with continuous monitoring of oligonucleotides by transfer of a nucleoside monophos- polymerase activity (Ferrari et al., 1999). Fluorescent phate from a nucleoside triphosphate (NTP) to the 3Ј assays to detect polymerase products are available hydroxyl of a growing oligonucleotide chain (Steitz, 1999). but require expensive detection equipment and often The driving force for this reaction is the cleavage of an the purification of the polymerase product. We seek to anhydride bond and the concomitant formation of inor- establish a convenient and sensitive colorimetric as- ganic pyrophosphate. The chemistry of oligonucleotide say for RNA polymerases that allows continuous mon- synthesis involves direct changes only at the ␣- and itoring of product formation. ␤-phosphoryl groups of the NTP, suggesting that struc- Several modifications of the base, ribose, and the tural modification of the ␥-phosphate of the NTP might ␥-phosphate gave NTPs that supported reduced, but not abolish its ability to function in the polymerase reac- detectable, RNA synthesis by the brome mosaic tion. In several cases, it has been found that DNA poly- virus (BMV) RNA-dependent RNA polymerase (RdRp). merases and reverse transcriptases may be sufficiently This latitude in polymerase activity led us to consider promiscuous to recognize and utilize nucleosides with attachment of a p-nitrophenyl (PNP) moiety to the modification at or in place of the ␥-position of the triphos- ␥-phosphates of GTP and ATP. This was accomplished phate moiety (Arzumanov et al., 1996). Little is known in a one-step chemical synthesis. PNP-modified about the ability of RNA polymerases to recognize NTPs nucleotides were found to be used in the synthesis of with modified ␥-phosphoryl groups. Such modified nu- RNA products by several RNA polymerases, including cleotides might be valuable for the development of sub- the RNA-dependent RNA polymerases from brome strates that display enhanced biological stability and mosaic virus and bovine viral diarrhea virus (BVDV) polymerase specificity as well as new types of RNA and the DNA-dependent RNA polymerases (DdRp) polymerase assays. from bacteriophage T7 and SP6. When the polymerase Currently, drugs affecting the activities of viral RNA reactions were performed in the presence of alkaline phosphatase, which converts p-nitrophenylpyrophosphate (PNP-PPi) to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was 1 Present address: ViroPharma Inc., Exton, Pennsylvania 19341. 2 To whom reprint requests should be addressed at Department of observed. This permits use of PNP–NTP analogues for Biology, Indiana University, 1001 E. Third St., Bloomington, IN 47405. continuous colorimetric monitoring of polymerase ac- Fax: (812) 855-6705. E-mail: [email protected]. tivity.

With the modified nucleotides, both UTP analogues were unable to support RNA synthesis (Fig. 1B, lanes 9 and 10), most likely due to the large sizes of the dye molecules attached to the uracil. MANT-GDP and MANT-GTP were found to substitute for the initiating GTP, resulting in low but detectable levels of synthesis (Fig. 1B, lanes 7 and 8). MANT-ATP, e-ATP, and NPE-ATP directed synthesis at ϳ20% of the level of normal ATP (Fig. 1B, lanes 4–6). These results indicate that a viral RNA replicase is able to accept a number of nucleotide analogues despite significant differences in the size and/or H-bonding capabilities of the analogues in comparison to normal ribonucleotides. While some of the modified nucleotides tested should allow for the detection of fluorescently labeled RNA prod- FIG. 1. Use of nucleotide analogues by BMV replicase. (A) The 33-nt RNA, Ϫ20/13, used by BMV replicase to direct synthesis of a 13-nt ucts, we were not able to detect the products due to the product, that is initiated from the arrow. (B) Products of the BMV lack of the suitable detector. Instead, we used this infor- replicase using nucleotides indicated above the autoradiograph. G, mation to design nucleotide analogues that can be easily GTP; A, ATP; U, UTP; and C*, [␣-32P]CTP; nucleotide triphosphate detected with a conventional laboratory spectrophotom- analogues include: ethano-ATP (e-ATP), 1-nitrophenyl-ethyl ester (NPE- eter. caged ATP), 2Ј-O-methylanthranilyl-modified ATP (Mant-ATP), Mant- GDP, Mant-GTP, Texas Red 5-UTP (TR-UTP), and tetramethylrhodamine (TMR-UTP). The lengths of replicase products are shown on the left. Synthesis of chromogenic nucleoside triphosphate While the same autoradiograph was used, the exposure time in lane 1 analogues used by RNA polymerases was shorter than that in lanes 2–11 so that the weak labeling in lanes 7 and 8 could be seen. Colorimetric assays for polymerase activity would per- mit monitoring of RNA synthesis in real time. The assay RESULTS we designed would use an NTP analogue with p-nitrophenol (PNP) attached to the ␥-phosphate of a NTP. To determine which nucleotide modifications are ac- Should an RNA polymerase use this NTP analogue, then ceptable to viral RNA polymerases, we used the BMV p-nitrophenylpyrophosphate (PNP-PPi) would be a by- RNA replicase. In the infection process, the BMV repli- product of phosphoryl transfer. In the presence of calf case needs to direct the de novo (primer-independent) intestinal alkaline phosphatase (CIP), PNP should be synthesis of three classes of RNAs: genomic minus- released and generate a yellow color in an alkaline strand, genomic plus-strand, and the subgenomic RNA4. solution (Fig. 2A). A similar two-step enzymatic reaction All three classes of RNAs can be produced in vitro with was previously used to assay ␣-amylase (Eisenthal and the subgenomic synthesis being the best characterized Danson, 1992). (Sivakumaran et al., 1999; Sun et al., 1996; Adkins et al., 1998). The correct initiation of BMV subgenomic RNA4 To synthesize a PNP–NTP analogue, a simple one- synthesis can be reproduced in vitro with a 33-nt RNA, step synthesis was adapted from procedures described -20/13, that is composed of the 20-nt promoter and a previously (Haystead et al., 1993). The synthesis and 13-nt template sequence (Siegel et al., 1997, 1998) (Fig. purification of PNP–GTP and PNP–ATP are described 1A). Products of 13 and 14 nt were also observed, with under Materials and Methods and shown in Fig. 2B. The the 14-nt product being previously demonstrated to con- final yield was reproducibly 65% of the input NTP. The 1 13 tain one nontemplated nucleotide added by the BMV structures of the final products were verified by H, C, 31 replicase (Fig. 1B). and P NMR (see Materials and Methods). Ribonucleoside triphosphate analogues tested are Experiments were performed to determine the concen- modified in the base [including 1, N6 ethano-ATP (e- tration of p-nitrophenylate needed to attain a significant ATP), Texas Red 5-UTP, and tetramethylrhodamine-6- absorbence at 405 nm. Para-nitrophenyl phosphate from UTP], the ribose (2Ј-O-methylanthranilyl-modified ATP, 100to1␮M were incubated with excess CIP for 30 min, GDP, and GTP), and at the ␥-phosphate [1-nitrophenyl- and absorbence was measured at 405 nm. A 10 ␮M ethyl ester (NPE-caged ATP)]. When substituting for solution of p-nitrophenylate gives an absorbence of 0.116 the corresponding ribonucleotide in a radiolabeled at 405 nm (data not shown). Thus, a 60-␮L reaction with RNA synthesis reaction, we found that reactions lack- 4 mM PNP–NTP (240 nmol) should result in an absor- ing ATP, UTP, or GTP resulted in no observable prod- bence of 0.464 at 405 nm if 1% of the input analogue was ucts (Fig. 1B, lanes 2 and 3, and results not shown). converted to p-nitrophenylate. COLORIMETRIC RNA POLYMERASE ASSAY 431

FIG. 2. Schematics of the colorimetric assay, PNP–NTP synthesis, and analysis. (A) Structures of the most relevant molecules in the colorimetric assay PNP–NTP, PNP-PPi, and PNP. In this assay, PNP–NTP is used in template-directed RNA synthesis by an RNA polymerase liberating the colorless side product, PNP-PPi. Calf intestinal phosphatase (CIP) then hydrolyzes PNP-PPi to the chromophore p-nitrophenylate, which absorbs maximally at 405 nM. (B) Synthesis of PNP–NTPs. A triethylamine (TEA) salt of a nucleotide triphosphate is attacked by dicyclohexyl carbodiimide (DCC), which induces a circularization of the phosphates. When p-nitrophenol is added, it attacks a phosphate, causing the linearization of the 1 13 phosphate groups and completing the synthesis of PNP–NTP. (C) H and C NMR spectra of 50 mM PNP–GTP in CD3OD.

Utilization of PNP-NTPs by various polymerases the BMV replicase to recognize moieties attached to the ␥-phosphate of NTPs (Fig. 1). To demonstrate whether the PNP–NTPs could be used The recombinant BVDV RdRp was previously demon- in nucleotidyl transfer reactions by RNA polymerases, strated to initiate RNA synthesis de novo from short reactions were performed with either PNP–ATP or PNP– GTP substituted for ATP and GTP, respectively, the re- templates (Kao et al., 1999). NS5B was able to use maining NTPs, and [␣-32P]CTP to allow visualization of PNP–GTP in a reaction where GTP was absent (W. Vas- the polymerase product. PNP–GTP was used at 200 ␮M, siliou, data not shown). In reactions where PNP–ATP while PNP–ATP was used at 100 ␮M (Kao and Sun, 1996; substituted for ATP, the expected 21-nt product was ob- ϳ Kao et al., 1999; Chamberlin and Ring, 1973). The higher served at 20% the amount seen in a reaction contain- GTP–PNP concentration was needed for the initiation of ing ATP (Fig. 3B, lanes 1–4). RNA synthesis by the BMV replicase (Kao et al., 1999). We tested the T7 RNA polymerase (T7 RPO) to deter- The reactions used a template for genomic plus-strand mine whether the ability to use ␥-phosphate modified synthesis that generates a 26-nt product and some in- NTP analogues was unique to RNA-dependent RNA syn- correctly terminated products of higher molecular weight thesis or is a common property of RNA polymerases. A (Sivakumaran et al., 1999). In the absence of either GTP DNA consisting of the T7 promoter followed by a 21-nt or PNP–GTP, no products were observed (Fig. 3A, lanes template was able to direct synthesis of a 21-nt product 4 and 5). The addition of PNP–GTP was able to restore in the presence of GTP (Fig. 3C, lane 1). When PNP–GTP synthesis to ϳ60% for the correct size product seen in was used in place of GTP, the expected 21-nt product the reaction with GTP (Fig. 3A, lanes 1–3). These results was detected at ϳ60% of the reaction containing GTP are consistent with the previous results with the ability of (Fig. 3C, lanes 2–4). When neither GTP nor PNP–GTP 432 VASSILIOU ET AL.

not degrade PNP–NTPs but will degrade normal nucleotides. The three templates contain 15 cytidylates (WV- 15C), 15 thymidylates (WV-15T), or an initiation cytidylate followed by 14 thymidylates (WV-C14T) (Fig. 4A). WV-15C can be used in a continuous colorimetric assay with PNP–GTP, while WV-15T, lacking an initiation cytidylate, would serve as a negative control. For assays with PNP– ATP, WV-C14T would provide a template cytidylate for initiation by the T7 RPO and can direct incorporation of AMPs. A guanine nucleoside will serve as the initiation nucleotide for the T7 RPO for this reaction (Martin and Coleman, 1989). A complete transcription reaction containing PNP– GTP and template WV-15C resulted in a linear increase in absorbence at 405 nm over a 2-h period (Fig. 4B). Several negative controls resulted in minimal absorbence changes, including a reaction containing WV-15T substituted for WV-15C, and reactions lacking T7 RPO, or Mg2ϩ (Fig. 4B). A significant increase in absorbence at

FIG. 3. PNP–NTP can be incorporated by three RNA polymerases. (A) Autoradiograph of BMV RdRp products from a template generating a 26-nt product. The nucleotides of interest are labeled at the top of the autoradiogram. Where used, GTP and PNP–GTP were at 200 ␮M. ⌽,a reaction lacking either nucleotides. (B) Autoradiograph of BVDV RdRp reaction products synthesized from a template generating a 21-nt product. Reactions in lanes 1–4 contained 100 ␮M ATP or PNP–ATP as indicated. ⌽, a reaction lacking both ATP and PNP-ATP. (C) Autoradio- graph of T7 RPO reaction products from a template generating a 21-nt product. Reactions in lanes 1–4 contained 200 ␮M GTP or PNP–GTP as indicated. ⌽, a reaction lacking both GTP and PNP–GTP. was present, the 21-nt product was no longer observed (Fig. 3C, lanes 5 and 6). The T7 RPO will also use the PNP–ATP analogue in transcription as will be shown later. These results indicate that PNP–NTPs are recognized and used in RNA synthesis by both DNA- and RNA-dependent RNA polymerases.

A continuous colorimetric assay for RNA polymerase activity FIG. 4. Homopolymeric templates and the continuous monitoring of To assess whether the PNP–NTPs can be used in a polymerase activity. (A) Homopolymeric DNA templates used in tran- continuous colorimetric assay, three DNAs consisting of scription by T7 RPO. (B) Absorbance changes over time due to PNP a double-stranded promoter followed by a homopoly- formation during transcription with PNP–GTP. Key components in each meric template sequence were made for transcription by reaction are listed to the right of the graph. A complete reaction 2ϩ F the T7 RPO (Milligan et al., 1987). The homopolymeric containing WV-15C, T7 RPO, and Mg ( ) is compared to reactions containing the inappropriate template WV-15T (Œ), lacking T7 RPO (ࡗ) template may allow the transcription to be performed or Mg2ϩ (▫). (C) Absorbance changes over time due to PNP formation with PNP–NTP as the sole nucleotide triphosphate, thus during transcription with PNP–ATP. The key components of the four permitting the presence of CIP in the reaction. CIP will reactions are indicated to the right of the graph. COLORIMETRIC RNA POLYMERASE ASSAY 433

over time was clearly dependent on the amount of polymerase in the reaction (Fig. 5B). Negative controls for these experiments included reactions with WV-15T, which should not direct the incorporation of GMP. These controls had only a slight increase in absorbence, most likely due the nonspecific degradation or the misincor- poration of PNP–NTP (Fig. 5B). Parallel reactions with WV-15T and PNP–ATP were performed. Again a more rapid increase in absorbence was observed with increasing amounts of polymerase (Fig. 5C). A reaction with WV-15C and PNP–ATP showed minimal absorbence (Fig. 5C). These results further confirm that the observed absorbence at 405 nm is dependent on the amount of RNA polymerase activity in the reaction.

Use of GTP vs PNP–GTP The preference of the T7 RPO for PNP–GTP or GTP was tested. Reactions were preformed with the ratios of FIG. 5. Effects of different amounts of CIP, T7 RPO, and different PNP–GTP to GTP ranging from 100:1 (4 mM: 0.04 mM) to PNP–NTP analogues on the colorimetric assay. (A) Changes in absor- 1:1 (4 mM to 4 mM), and A values were compared to bance with 10, 5, and 2.5 units of CIP. A scan of one these reactions 405 after CIP addition is shown in the inset to demonstrate that maximum reactions containing only PNP–GTP at 4 mM. Increasing absorbance is at 405 nm. (B) The effects of T7 RPO at 22, 11, and 5.5 use of GTP should reduce utilization of PNP–GTP, result- ␮g/60 ␮L on colorimetric assays with 4 mM PNP–GTP at 0.5 and 2.0 h ing in a decrease of absorbence at 405 nm. When PNP– after the addition of polymerase. (C) Use of PNP–ATP in a transcription GTP was present at a concentration 50-fold higher than reaction with T7 RNA polymerase. Use of template WV-C14T allows GTP, little effect on absorbence was observed relative to initiation of RNA synthesis while WV-15C should not. PNP–GTP alone (Fig. 6). However as the ratio of PNP– Յ GTP to GTP was 25:1, a progressive decrease in A405 405 nm over time was observed only for reactions con- was observed. These results indicated, not surprisingly, taining PNP–ATP with WV-C14T but only in the presence that T7 RPO would selectively use GTP rather than PNP– 2ϩ GTP. Preference for normal NTPs was also observed of T7 RPO and Mg . Furthermore, no significant change ␥ in absorbence was seen when WV-15C was incubated with other -phosphate modified NTP analogues used by with T7 RPO and PNP–ATP (Fig. 4C). These results dem- the BMV RNA replicase (Fig. 1) and in the radiolabel onstrate that the T7 RNA polymerase can use specific incorporation assay (Fig. 3). PNP–NTPs in transcription reactions, in the presence To further quantify the difference in the use of GTP and CIP, to continuously monitor transcription. PNP–GTP, an apparent Km of PNP-GTP was determined using the colorimetric assay with variable PNP–GTP con- The dependence of color formation on polymerase Ϯ and CIP concentrations was tested. For color production centrations. A preliminary Km value of 2.8 0.43 mM was to be indicative of polymerase activity, the conversion of PNP-PPi to PNP by CIP should not be rate limiting. Thus CIP was added at 10, 5, and 2.5 units after transcriptions had taken place for 2 h with T7 RPO, PNP–GTP, and WV-15C. All three CIP concentrations resulted in maximal absorbence within 2.5 min, with a peak absorbence at 405 nm (Fig. 5A and inset). The action of CIP was so rapid that the absorbence increased as soon enzyme was added, hence the variations at the 0 time point for the three CIP concentrations. These results demonstrate that CIP activity was not rate limiting in the polymerase assays; thus change in absorbence is primarily determined by RNA polymerase activity. To demonstrate further that polymerase activity is the primary determinant of the observed absorbence, sev- FIG. 6. Preference of T7 RPO for PNP–GTP and GTP. Absorbances for transcription reactions with T7 RPO, WV-15C, and various ratios of eral concentrations of T7 RPO were tested. Reactions PNP–GTP to GTP. Each reaction was allowed to incubate at 37° C for with WV-15C and PNP–GTP were preformed with T7 RPO 2 h and contained T7 RPO and 4 mM PNP–GTP with GTP concentra- present at 22, 11, and 5.5 ␮g. The increase in absorbence tions at 0, 0.04, 0.08, 0.16, 0.4, 0.8, and 4 mM. 434 VASSILIOU ET AL.

FIG. 8. Continuous colorimetric assay with 10% plasma. Key compo- FIG. 7. Colorimetric assays with plasmid templates. T7 RPO tran- nents in the transcription reaction are listed to the right of the graph. scriptions with 4 mM each of ATP, UTP CTP, PNP–GTP and either a The reactions with WV-15C (F) should direct initiation by the T7 RPO plasmid containing a T7 promoter (pBSII KSϪ) or lacking one (pUC18). while WV-15T (▫) should not. To test for stability of PNP–GTP, reactions Numbers on the horizontal axis indicate times at which CIP was added were preformed with CIP but no T7 RPO (ࡗ) and PNP–GTP with to the reaction. The absorbance was measured 5 min after the addition plasma alone (Œ). of CIP. obtained for PNP–GTP and of 1.5 mM for PNP–ATP (data Colorimetric monitoring of SP6 polymerase activity not shown). These values indicate that millimolar was then tested. The reactions contained the SP6 poly- amounts of the NTP analogues are needed in a colori- merase and either the plasmid pTRI, possessing an SP6 metric polymerase assay. For comparison, reported promoter, or the plasmid pUC18, which lacks this pro- value of the T7RPO for GTP and ATP are 0.16 and 0.047 moter (Table 1). An increase in absorbence at 405 nm mM (Chamberlin and Ring, 1973). over time is observed in the reaction with SP6 RPO and pTRI, while absorbence remains minimal in the reaction Use of PNP–GTP in transcription from plasmids that lacked SP6 RPO or had SP6 RPO along with pUC18. The increase in absorbence above background was be- PNP–GTP can be utilized by RNA polymerases using tween 3.8- and 5-fold (Table 1). These results demon- homopolymeric templates. To test PNP–NTPs with more strate that our colorimetric assay can be used to follow biologically relevant templates, plasmids pBSII KSϪ and the activity of the T7 and SP6 RNA polymerases during pUC18 were used. pBSII KSϪ contains a T7 promoter synthesis from nonhomopolymeric templates. while pUC18 does not. Reactions with plasmid templates contained ATP, CTP, UTP as well as PNP–GTP each at 4 Continuous colorimetric assays and stability of PNP– mM. Reactions were allowed to proceed for a desired GTP in plasma period of time, after which CIP was added and A405 was determined 5 min later. A mixture of the components not To test the continuous colorimetric assay and the incubated for any period of time, thus preventing tran- stability of PNP–GTP in a more complex environment, scription by RNA polymerase, and treated with CIP was transcriptions were conducted with the addition of used as a blank. An increase of A405 over time was plasma to 10% final concentration. A complete transcrip- observed in the reaction with pBSII KSϪ, while absor- tion reaction containing WV-15C and PNP–GTP with 10% bence remains minimal in the reaction with pUC18 plasma showed an increase in absorbence at 405 nm up (Fig. 7). to 4 h (Fig. 8), while the control reaction containing the

TABLE 1 Colorimetric Detection of SP6 RNA Polymerase Activitya

A405 SP6 ϩ pTRI Time (h) ⌽ϩpTRI SP6 ϩ pUC18 SP6 ϩ pTRI SP6 ϩ pUC18

0— — — — 1 0.029 Ϯ 0.017 0.031 Ϯ 0.022 0.126 Ϯ 0.015 4.0 2 0.051 Ϯ 0.018 0.056 Ϯ 0.013 0.215 Ϯ 0.021 3.8 3 0.049 Ϯ 0.011 0.063 Ϯ 0.019 0.334 Ϯ 0.027 5.3

a SP6 RNA polymerase was tested in colorimetric assays with 7.5 mM each of ATP, UTP, CTP, and PNP-GTP. Absorbance values for reactions lacking SP6 RPO (⌽) or lacking an appropriate promoter in the plasmid, pUC18, are shown in the first two columns. Absorbance values for reactions with SP6 RPO and a plasmid containing an SP6 promoter, pTRI, are shown in the third column. The last column gives the fold of synthesis above background. Values are expressed as one standard deviation from the mean. COLORIMETRIC RNA POLYMERASE ASSAY 435 template WV-15T had a minimal absorbence over the are highly similar members of the Flaviviridae virus fam- same time period, as did reactions containing only PNP– ily. A continuous colorimetric assay with a nonhomopoly- GTP with or without CIP (Fig. 8). Results here demon- meric template will be possible if all substrate nucleo- strate that PNP–GTP is stable in 10% plasma and can be tides were PNP–NTPs. used to assay biological relevant samples. The addition of moieties that can generate fluorescence after polymerization should greatly enhance the DISCUSSION sensitivity of detection because fluorescence detection is more sensitive than spectrophotometric measure- The use of ␥-phosphate-modified rNTPs by RNA poly- ments (Guilbault, 1990). Additional modifications that merase and RdRps demonstrates a flexibility in poly- confer polymerase specificity, altered detection, or may merase-substrate interaction. Previously Arzumanov et be used as prodrugs could be made using the same al. found that DNA-dependent DNA polymerases and chemistry. Deoxynucleotides with similar modifications reverse transcriptase can accept modifications at the at the ␥-phosphate may allow colorimetric assays with ␥-phosphate; 2Ј dTTP with a 5Ј-␥-phenyl or a 5Ј-␥- anilido DdRps or RTs. moiety were used for DNA synthesis by DNA poly- ␣ ␤ merases and , while the same modifications to the MATERIALS AND METHODS ␥-phosphate of azido-2Ј,3Ј-dideoxythymidine triphosphate (AZTTP) resulted in nucleotides that were incorpo- Materials rated into DNA by the HIV reverse transcriptase and Nucleoside triphosphate analogues were from Molec- caused the termination of DNA synthesis (Arzumanov et ular Probes (Eugene, OR). Ribonucleotides were from al., 1996). Amersham Pharmacia Biotech. except the guanosine Chromogenic reagents have previously been used to 5Ј-triphosphate, and adenosine 5Ј-triphosphate used in measure gene expression levels and specific enzymatic chemical synthesis, which were from Sigma. N,NЈ-dicy- activity (Alam and Cook, 1990; Bergmeyer, 1986). Here clohexylcarbodiimide was from Acros, and p-nitrophenol we describe a convenient colorimetric assay for moni- was from J.T. Baker Inc. Deoxyoligonucleotides were toring RNA polymerase activity. Using nucleotide ana- synthesized by Operon Technologies. Plasmids pUC18 logues with a p-nitrophenyl moiety attached to the and pBSIIKSϪ were from Gibco BRL and Stratagene, ␥-phosphate we were able to detect the activities of two respectively. The RNA template for BVDV NS5B radiola- viral RdRps and two DdRps by monitoring absorbence of beled assays was chemically synthesized by Oligos Etc. the reaction at 405 nm. Synthesis of PNP–NTPs is an The RNA template for BMV replicase radiolabled assays inexpensive, simple one-step process with well-estab- was synthesized as previously described (Sivakumaran lished purification protocols. For the T7 RPO, K values m et al., 1999). for PNP–GTP and PNP–ATP were 2.8 and 1.5 mM, respectively. Continuous monitoring of T7 RPO activity was Synthesis of PNP–NTP analogues achieved with homopolymeric templates even in the presence of 10% plasma. The colorimetric assay could Guanosine-5Ј-(␥-4-nitrophenyl) triphosphate (PNP– be developed for high throughput assays for polymerase GTP) and adenosine-5Ј-(␥-4-nitrophenyl) triphosphate inhibitors and for the analysis of the mechanism of RNA (PNP–ATP) were synthesized using a modified procedure synthesis. previously described (Haystead et al., 1993). A standard It is important to note that this assay does not directly reaction consists of 0.3 mmol monosodium salt of GTP or detect polymerase products. Instead, CIP hydrolyzes the ATP dissolved in 2 mL of 0.1 M triethylamine bicarbonate PNP-PPi, which is liberated during RNA synthesis. Hy- (solution A) and passed over a 20 ϫ 1 cm column of drolysis of PNP-PPi yields the spectrophotometrically cation exchange resin (Ag 50W-X8, Bio-Rad) equilibrated detectable chromophore PNP. This indirect method of in solution A. The column was washed with 25–50 mL of determining polymerase activity is valid because there is solution A, and the eluate was concentrated to dryness. minimal PNP production when the reaction lacked poly- Five milliliters of anhydrous methanol was added to the merase, an appropriate template, or Mg2ϩ (Fig. 4B). Also residue and evaporated, the procedure was repeated the reaction was dependent on the amount of polymer- twice more, and the residue was dried under high vac- ase and not limited by the activity of CIP (Fig. 5). uum for 30 min. The residue was dissolved in 5 mL of Our continuous colorimetric assay was performed with dimethyl sulfoxide, then 3 mmol of dicyclohexyl carbodi- homopolymeric templates. Characterizations of BVDV imide (DCC) was added and the mixture was stirred RdRp, Rabbit Hemorrhagic Disease Virus (RHDV) RdRp, under nitrogen overnight. Six millimoles of p-nitrophenol and Hepatitis C Virus (HCV) RdRp also used homopoly- followed by 6 mmol of triethylamine were added and meric templates in radiolabeled assays (Sun et al., 1996; stirred under nitrogen overnight. The solution then was Zhong et al., 1998; Vasquez et al., 1998). We expect the diluted with 20 mL of water and acidified to pH 5 with HCV RdRp to use PNP–NTPs because BVDV and HCV acetic acid. Excess p-nitrophenol was extracted from the 436 VASSILIOU ET AL.

aqueous phase with diethyl ether (50 mL ϫ 4) in a Ultramicro quartz cuvette (accurate with 50-␮L volume separatory funnel. The water phase was reduced by samples) at 405 nm using a spectrophotometer (Spec- evaporation to 5 mL and loaded onto an 8 ϫ 2.5 cm tronic Genesys 5). DEAE anion exchange column equilibrated with solution Variations of the standard assay occurred when the A. The column was washed with 500 mL of solution A, effect of T7 RPO was tested using 22, 11, and 5.5 ␮gofT7 and the product was eluted with 0.5 M triethylamine RPO per 60-␮L aliquot. When CIP was tested 10, 5, and bicarbonate. Fractions absorbing at 259 nm were pooled 2.5 units/60 ␮L aliquot were added after the reaction had and the solvent evaporated. The residue was dissolved proceeded for2hat37°C. Preference assays for GTP– in 2 mL of water and treated with 100 units of calf PNP and GTP were conducted under standard condi- intestinal phosphatase (CIP, New England Biolabs) for tions except that 4 mM PNP–GTP was incubated with a 2 h at room temperature and purified again by DEAE range of GTP concentrations from 0.04 to 4 mM. Assays anion exchange. Fractions absorbing at 259 nm were with plasmids contained pUC18 or pBSII KSϪ at 12.5 ␮ pooled, evaporated, and then dissolved in sufficient H2O ng/ L. In these reactions, 4 mM each of UTP, CTP, ATP, to make a 100 mM solution. PNP–NTP yield was deter- PNP–GTP was present, and CIP was added after the mined by spectrophotometry. The product purity and reaction had proceeded for a desired time. composition were verified by 31P, 13C, and 1H NMR with a Transcriptions with SP6 RNA polymerase were per- VXR400 and Gem300 spectrometer, and found to be 95%. formed using a Megascript SP6 kit (Ambion) following The NMR results to verify the structure of PNP–GTP the manufacturer’s protocol, except that 7.5 mM PNP– 1 were as follows. PNP–GTP- H NMR (400 MHz, CD3OD, GTP was substituted for the GTP. The template used was ␦, ppm): 8.15 (d, 2H, aromatic); 8.05 (s, 1H, H-8); 7.52 (d, pTRI-xef linearized plasmid at 20 ng/␮L. Aliquots (60 ␮L) ϭ Ј 2H, aromatic); 5.83 (d, 1H, J1Ј,2Ј 6 Hz, H-1 ); 4.73 (t, 1H, of the transcription were removed after a desired period Ј ϭ ϭ Ј H-4 ); 4.48 (dd, 1H, J3Ј,2Ј 3.2 Hz, J3Ј,4Ј 4.8 Hz, H-3 ); of time and treated with 5 units of CIP for 5 min before ϭ Ј 4.29–4.25 (m, 2H, J5Јa,5Јb 15.6 Hz, H-5 a,b); 4.18–4.16 (m, absorbence was determined. Ј 13 ␦ 1H, H-2 ). C NMR (100 MHz, CD3OD, , ppm): 158.7, ϭ 158.4, 154.4, 152.1, 143.3, 137.1, 125.0, 121.1 (d, JCP 5.3 Km determination ϭ 31 Hz), 116.7, 88.0, (d, JCP 9.1 Hz), 74.1, 71.0, 65.5. P (121 ␦ Ϫ ϭ ␥Ϫ Ϫ Absorbence at 405 nm was measured for standard T7 MHz, CD3OD, , ppm): 10.6 (d, J 18.3 Hz, P); 17.0 ϭ ␣ Ϫ ϭ ϭ RPO colorimetric assays incubated at 37°C for 2.5 h with (d, J 18. 2Hz, -P); 22.4 (dd, J␤ ,␥ 18.3 Hz, J␤ ,␣ 18.2 Hz, ␤-P). concentrations of PNP–NTP ranging from 0.5 to 10 mM. A405 values were then plotted against the PNP–NTP con- Radiolabeled polymerase assays centration using the program Enzyme Kinetics (version 1.0 b, Dogstar software Bloomington, IN). BMV RdRp was prepared from infected barley and assayed as previously described (Sun et al., 1996). BVDV ACKNOWLEDGMENTS NS5B was prepared from recombinant baculovirus-infected Sf9 cells and assayed as described (Zhong et al., We are grateful to David Daleke for providing human plasma, mem- 1998; Kao et al., 1999). Synthesis was quantified relative bers of the Widlanski laboratory for technical assistance during the to reactions containing only nonmodified NTPs. All ex- synthesis of PNP–NTPs, and members of the IU Cereal Killer group for helpful discussions during the course of this work. Funding was pro- periments were assayed with duplicate samples re- vided in by the National Science Foundation Grant 980700 and United peated in at least one other independent assay. States Department of Agriculture Grant 9702126 to C.K. and National T7 RPO was prepared and assayed as described, Cancer Institute Grant 1 F32 CA 77883–01 to J.B.E. except that reactions were spiked with 250 nM ␣ 32 [ - P]CTP (400 Ci/mmol, 10 mC/mL, Amersham) (Grod- REFERENCES berg and Dunn, 1988). Adkins, S., Stawicki S. S., Faurote, G., Siegel, R. W., and Kao, C. C. Colorimetric polymerase assay (1998). 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