Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs
© Sun and Wang; licensee BioMed Central Ltd. 2013
Received: 11 April 2013
Accepted: 24 July 2013
Published: 6 August 2013
Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.
Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.
We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.
Mycoplasmas are wall-less, gram-positive bacteria and are pathogenic to humans, animals, and plants . Mycoplasma pneumoniae (Mpn) is a human pathogen and causes acute and chronic diseases at multiple sites. Respiratory diseases dominate and account for approximately 30% of cases of community-acquired pneumonia. Mpn may also be a direct cause or significant cofactor in many extrapulmonary diseases including rheumatoid arthritis, and central nerve system manifestations such as encephalitis, aseptic meningitis, acute transverse myelitis, stroke, chronic fatigue, and polyradiculopathy [1–3]. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin, and macrolides are the treatment of choice.
Increased incidences/epidemics of Mpn infections have been reported in Scandinavian countries, France, Scotland, and Israel from 2010 to 2012 [4, 5]. In most cases, the infected individuals did not need medical attention. However, approximately 10% of the patients developed pneumonia and antibiotic treatment was needed. In severe cases, hospitalization was required and there were lethal cases when patients were infected by macrolide-resistant Mpn strains [6, 7]. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. In Japan and China, approximately 90% of the isolates are resistant to erythromycin or azithromycin, especially among pediatric patients [8–12]. This limits treatment options for patients with severe Mycoplasma pneumonia caused by macrolide resistant Mpn strains. Therefore, new antibiotics are needed.
Nucleotides are not only the building blocks of DNA and RNA, but also regulatory factors in diverse cellular metabolic pathways, and therefore, inhibition of enzymes in this pathway will cause nucleotide pool imbalance, which will inhibit DNA and RNA synthesis and lead to cell death. When transported into and metabolized by cells, nucleoside analogs can interfere with metabolism of natural nucleotides and/or DNA and RNA synthesis. An example of this type of antibiotic is sulphonamides such as sulfamethoxazole that target dihydropteroate synthetase in the folic acid biosynthesis pathway, and inhibition of folic acid biosynthesis leads to impaired purine and pyrimidine nucleotide biosynthesis . Another example is thymidylate synthase (TS) inhibitors such as Ralitrexed and 5-fluorouracil used as anticancer drugs [14, 15]. Today more than 50% of the United States Food and Drug Administration (FDA)-approved anticancer and antiviral drugs are nucleoside and nucleobase analogs. The most successful reports since the 1970s, using nucleoside analogs as drugs, were for the treatment of herpes viral infections by acyclic guanosine analogs such as acyclovir, and HIV infection by nucleoside analogs such as Zidovudine or Lamivudine in combination with protease inhibitors i.e., highly active antiretroviral therapy [16, 17].
Compared to other antibacterial compounds, most nucleoside and nucleobase analogs used in anticancer and antiviral therapy have narrow therapeutic index and adverse side effects, with the exception of acyclic guanosine analogs used in the treatment of herpes viral infection. These adverse effects limit their use in the treatment of bacterial infections. In recent years efforts have been made to develop nucleoside analog based antibiotics, taking the advantage of structural differences in target enzymes between bacteria and their host to design and select specific inhibitors that will selectively kill the bacteria and spare the host [18–23]. For example, selective thymidylate kinase inhibitors have been developed and showed potent inhibitory effect in vivo against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus[22, 23]. Toxicity or side effect of these thymidylate kinase inhibitors to humans remains to be seen.
Mycoplasmas, in general, depend on exogenous supply of precursors for their nucleotide biosynthesis because they lack the de novo synthesis of purine and pyrimidine bases. Nucleosides and deoxynucleosides are efficiently taken up and phosphorylated to their respective nucleotides by deoxynucleoside kinases such as thymidine kinase (TK) and deoxyadenosine kinase. Nucleobases are salvaged through hypoxanthine guanine phosphoribosyltransferase (HPRT), adenine phosphoribosyltransferase (APRT) and uracil phosphoribosyltransferase (UPRT) systems [24–32]. Of a total of 17 enzymes in nucleotide biosynthesis identified in the Mpn genome, 15 are essential. Enzymes mentioned above, TK, HPRT, APRT and UPRT are essential for Mpn survival while thymidylate synthase (TS), an enzyme catalyses the reductive synthesis of thymidylate from uridylate, is not since thyA mutant Mpn strain that lacks TS is viable [31, 33, 34].
In this study, 30 FDA-approved nucleoside and nucleobase analogs that are anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth. Seven analogs showed potent inhibitory effects on Mpn growth at clinically achievable plasma concentrations. Among them, 6-thioguanine (6-GT) inhibited Mpn growth with a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1. To investigate the mechanism of action of these drugs, we studied the effects of these analogs on uptake and metabolism of natural nucleoside and nucleobases by using tritium labelled natural substrates. Furthermore Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned and expressed, and the recombinant enzyme was purified and characterized using tritium labelled hypoxanthine and guanine as substrates, and 6-thiuoguanine and other purine analogs as inhibitors. The role of thymidine kinase in the inhibitory effect of trifluorothymidine against Mpn growth was also investigated.
Inhibition of Mpn growth by nucleoside and nucleobase analogs
Inhibition of M. pneumoniae growth by nucleoside and nucleobase analogs*
Wild type MIC (μg ml-1)
thyA mutant MIC (μg ml-1)
For most compounds, the inhibitory effects were similar between the wild type and the thyA mutant Mpn strains, however differences between the two Mpn strains were also observed. For example, gancyclovir inhibited wild type Mpn but not the thyA mutant, whereas valacyclovir did not inhibit Mpn growth. Ribavirin and pentoxifylline inhibited wild type Mpn but not the thyA mutant. Among the 5-halogenated pyrimidine analogs, most of them inhibited both the wild type and the thyA mutant strain, but 5-iododeoxyuridine only inhibited the wild type Mpn growth (Table 1).
Uptake and metabolism of natural nucleosides and nucleobases in the presence of analogs
Inhibition of tritium labelled natural nucleoside and nucleobase uptake and metabolism by selected analogs*
0.005 ± 0.0004
Up-regulation of Mpn TK activity by TFT
Expression, purification, and characterization of HPRT
The purine analog 6-TG strongly inhibited Mpn growth, which promoted further investigation of potential targets of this compound. HPRT is the first enzyme in the salvage pathway of purine bases for nucleotide biosynthesis, and is the enzyme responsible for metabolizing 6-TG in human patients treated with this drug . Mpn HPRT (MPN672) consists of 175 amino acids and shares 29% sequence identity to human HPRT. Mpn HPRT cDNA was cloned and expressed in E. coli. Recombinant Mpn HPRT was expressed as an N-terminal fusion protein with a 6 × histidine tag and a tobacco etch virus (TEV) cleavage site at the N-terminus, and was purified to >98% purity by metal affinity chromatography, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (data not shown).
Inhibition of Mpn and human HPRT with 6-TG and other purine analogs
Inhibition of Mpn and human HPRT by purine analogs *
3.5 ± 0.5
20.5 ± 6.5
3.16 ± 0.2
39.8 ± 4
89.7 ± 14.5
22.5 ± 3.6
281.8 ± 21
25.1 ± 3
1585 ± 134
2511 ± 156
Trifluorothymidine (TFT) as substrate for human thymidine kinase (TK) 1, TK2, and Ureaplasma TK
Kinetic parameters of trifluorothymidine with purified recombinant human TK1, TK2, and Ureaplasma TK*
5.9 ± 1.7
0.043 ± 0.003
7.3 ± 1.8
8.8 ± 3.8
0.026 ± 0.003
3.0 ± 0.8
9.9 ± 5.2
0.055 ± 0.008
5.6 ± 1.5
Inhibition of human TK1, TK2, and Ureaplasma and Mpn TK by TFT and 5FdU
IC 50 values (μM) of trifluorothymidine (TFT) and 5-fluorodeoxyuridine (5FdU) with purified recombinant human TK1 and TK2, Ureaplasma TK, and Mpn extracts *
9.7 ± 3.2
75.9 ± 2.6
80 ± 5.6
158.5 ± 2.7
12.0 ± 4.2
1000 ± 13.3
9.1 ± 2.9
Our screening of 30 FDA-approved anticancer and antiviral nucleoside analogs revealed seven potent inhibitors of Mpn growth with MIC values at clinically achievable plasma concentrations. Nucleoside and nucleobase analogs used in anticancer and antiviral therapy are prodrugs. In order to exert their therapeutic potential they have to compete with natural substrates for uptake (e.g. transport across plasma membrane) and metabolism (e.g. enzymes that activate them to their active forms). Once phosphorylated these analogs are trapped inside the cells and further metabolized to their active form by cellular enzymes, therefore, competition/inhibition of enzymes (e.g. TK or HPRT) in the initial phosphorylation step would also affect the uptake and metabolism of these compounds, and thus their cytotoxic effect (Figure 4). As shown in Table 2, dipyridamole and 6-TG inhibited Hx and Gua uptake and metabolism but not Ade or Ura, suggesting that HPRT may be an immediate target. Pyrimidine nucleoside analogs e.g. TFT, 5FdU and dFdC affected the uptake and metabolism of all radiolabeled substrates, indicating that multiple enzymes/steps are involved and the extent of uptake of specific nucleoside or nucleobase is determined by activities of these metabolic enzymes (Figure 4).
The 6-TG inhibited Mpn growth with MIC value of 0.20 μg ml-1, which is equivalent to tetracycline (MIC = 0.1 μg ml-1). However, 6-MP, a 6-TG analog did not inhibit Mpn growth. Neither theophylline, 7-(2, 3-dihydroxypropyl) theophylline, allopurinol, nor caffeine inhibited Mpn growth. 6-TG strongly inhibited uptake and incorporation of nucleotides derived from Hx and Gua into DNA and RNA, indicating that the observed inhibition by 6-TG was both at the level of transport and metabolism. It is noteworthy that the uptake/metabolism of Hx and Gua was inhibited by all the analogs used.
Thiopurines, especially mercaptopurines, are the first line drugs for the treatment of acute leukemia since the 1950s. They are also used in the treatment of inflammatory bowel disease . The 6-TG and 6-MP exert their cytotoxicity through incorporation into DNA as deoxy-6-thioguanosine. These thiopurines are metabolized to deoxy-6-thioguanosine triphosphate via the purine salvage pathway initiated by HPRT (Figure 4). Thiopurine methyl transferase is a key enzyme in converting mercaptopurine to its cytotoxic metabolites, which can either inhibit purine nucleotide biosynthesis or incorporate into DNA or RNA, causing DNA damage and cell death . Mpn does not possess the essential enzymes, inosine monophosphate dehydrogenase and thiopurine methyl transferase, to convert mercaptopurine to the cytotoxic thioguanine nucleotides, the respective methyl thiopurine nucleotides. This may explain why 6-MP did not inhibit Mpn growth.
To further investigate the mechanism by which 6-TG inhibited Mpn growth, Mpn HPRT was expressed, purified, and characterized. Both Hx and Gua are good substrates for the enzyme and the Vmax values for these substrates are in the same order of magnitude as the human enzyme . In humans, the plasma concentrations of Hx and Gua are approximately 172 μM and 97 μM , which is close to the Km and S0.5 values of Mpn HPRT with Hx and Gua. These results suggest that Mpn HPRT is capable of efficiently salvaging both Hx and Gua. In addition, Mpn HPRT showed positive cooperativity with Gua, indicating that at higher Gua concentration the enzyme utilizes Gua better.
6-TG and 6-MP are structural analogs. The observed significant differences in their inhibitory effects with Mpn and human HPRT suggest that there are structural differences in binding of these two compounds to the respective HPRTs in their active sites. These differences could be used in future design of Mycoplasma specific inhibitors. HPRT has been suggested as a target for anti-parasite drug development and new compounds have been developed .
Halogenated pyrimidine analogs such as 5FdU inhibited Mpn and Ureaplasma growth, as reported in our earlier studies [30, 35]. Others have shown that TFT and 5FdU inhibited Cryptosporidium growth , and gemcitabine inhibited Staphylococcus aureus growth in vitro and in animal models . In this study, several halogenated pyrimidine analogs inhibited Mpn growth, and TFT and dFdC were more potent than 5FdU. The mechanism of inhibition by dFdC is most likely due to inhibition of ribonucleotide reductase and incorporation into DNA by dFdC metabolites (Figure 4). We did not observe significant differences in the inhibitory effects between the wild type and the thyA mutant strains, suggesting that TS activity is not required for toxicity of these compounds to Mpn.
Mycoplasma TK is an essential enzyme while TS is not [31, 33, 34]. The expression of TK in Mpn was correlated with Mpn growth and DNA synthesis, and upregulation of TK activity was observed in an Mpn strain lacking TS activity . The phosphorylated products of TFT and 5FdU by TK irreversibly inhibit TS activity via covalent binding to the enzyme, and down regulation of TS activity leads to upregulation of TK activity, similar to what was observed with the thyA mutant . Increased salvage of dT due to the induction of TK activity leads to higher level of dTTP, an allosteric regulator of purine nucleotide reduction by ribonucleotide reductase. Inhibition of ribonucleotide reductase activity by high level of dTTP led to decreased uptake and incorporation of labelled nucleobases as shown in this study, which may result in imbalance in nucleotide pools. In addition, high TK activity facilitates the phosphorylation of TFT and 5FdU and accumulation of TFT-TP and 5FdUTP that may affect the integrity of DNA and lead eventually to cell death (Figure 4). The fact that both TFT and 5FdU inhibited the growth of both wild type and the thyA mutant strain to the same extent, and the TK activity is upregulated by TFT and 5FdU, suggests that TK plays an important role in growth inhibition observed with these compounds.
In this study we have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the plausible mechanism of growth inhibition by these analogs are due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. We should keep in mind that the analogs used in this study are potent anticancer and antiviral drugs and most of them have diverse adverse side effect in humans and therefore, they may not be suitable for treatment of a mild Mpn infection. However, the results obtained with these analogs may be used as leads in the design of Mycoplasma specific inhibitors, substrates, or non-substrate inhibitors for the target enzymes in order to reduce the risk of host cell toxicity. More work regarding the mechanism of action of these drugs is needed. This study has provided the basis for future development of antibiotics against Mycoplasma or other bacteria.
Radiolabelled substances: [3H]-hypoxanthine ([3H]-Hx, 13.8 Ci/mmol), [3H]-thymidine ([3H]-dT, 20 Ci/mmol), [γ-32P]-ATP (2000 Ci/mmol), [3H]-Uracil ([3H]-Ura, 31.65 Ci/mmol), and [3H]-adenine ([3H]-Ade, 27.2 Ci/mmol) were purchased from PerkinElmer. [3H]-guanine ([3H]-Gua, 10.7 Ci/mmol) and [5-3H]-deoxyuridine 5’-monophosphate ([3H]-dUMP, 27 Ci/mmol) were from Moravek Biochemicals, Inc. The nucleoside and nucleobase analogs library  was kindly provided by Professor Pär Nordlund, from the Karolinska Institute, Stockholm, Sweden. Phosphoribosyl pyrophosphate (PRPP), dipyridamole, tetracycline, and nonradioactive Hx and Gua were from Sigma-Aldrich.
Mpn culture, and the effects of nucleoside and nucleobase analogs on growth and metabolism
Nucleoside and nucleobase analogs were dissolved in dimethyl sulfoxide (DMSO) as stock solutions and diluted with Mpn culture medium to the desired concentration immediately prior to use. The DMSO concentration in the final dilution was < 1%, which would not interfere with Mpn growth.
Mpn laboratory strain M129 wild type and a thyA mutant strain  were used in this study. Mpn was cultured at 37°C in a CO2 incubator using 75 cm2 tissue culture flasks containing 50 ml Hayflick’s medium, and harvested at day 4 when the medium color change was observed . The cells were harvested and the pellet was resuspended in 6 ml fresh medium and the cfu/ml was determined by serial dilution (10-fold) and plating on broth agar plate. Colonies was counted and cfu/ml was calculated.
Inhibition studies were performed in 96-well plates containing 200 μl Mpn culture (approximately 106 cfu ml-1) in Hayflick’s medium and 200 μl each compound in series dilutions (2-fold) with the growth medium, with three to four replicas. The plates were sealed with clear adhesive sheets and incubated at 37°C incubator. Absorbance ratio at 450 nm and 560 nm was used as Mpn growth index, which was measured daily, and by visual detection for at least 8 days, as previously described . In the absence of inhibitor, the culture medium turned yellow on day 4. Controls were cultured in the presence of 2 μg/ml tetracycline, which showed no growth for up to 8 days. Medium was placed in four wells per plate for controls, which showed no color change during the incubation period. The MICs (minimal inhibitory concentration required to inhibit Mpn growth to 90%) were determined as the lowest concentration at which the growth index was ≈ 10% of the control values (at the time when the control culture medium color turned yellow), essentially as described .
Nucleoside and nucleobase uptake and metabolism was done with the wild type strain, which was cultured in 25 cm2 tissue culture flasks, inoculated with 1 ml stock culture (1 × 108 cfu/ml) Mpn, in the presence of tritium labeled dT, Hx, Gua, Ade or Ura (1 μCi ml-1) and the presence or absence of nucleoside and nucleobase analogs (10 μM) and incubated at 37°C for 70 hours. The cells were harvested and analyzed essentially as described . Analogs used were dipyridamole, 6-thioguanine (6-TG), trifluorothymidine (TFT), gemcitabine (dFdC) and 5-fluorodeoxyuridine (5FdU). Total uptake is the percent of radioactivity recovered in the cells divided by total radioactivity added to the growth medium. Percent of acid insoluble (radioactivity found in DNA and RNA) was also calculated . These experiments were done more than three times and data are given as mean ± SD.
To determine the effect of TFT on TK and TS activity, Mpn wild type cells were cultured in 75 cm2 tissue culture flasks containing 50 ml medium, inoculated with 3 ml of stock culture (1 × 109 cfu/ml), in the presence of [3H]-dT (1 μCi ml-1) and different concentrations of TFT. After 70 hours at 37°C the cultures were harvested and divided to two aliquots, one was used to determine total uptake/metabolism of radiolabeled dT and total proteins were extracted from the other aliquot and used to measure TK and TS activity using [3H]-dT and [5-3H]-dUMP as substrates .
Expression and purification of recombinant Mpn HPRT
The Mpn HPRT gene (MPN672) coding sequence was codon optimized for expression of the recombinant protein in E. coli, by using the Proprietary OptimumGene™ codon optimization technology combined with gene synthesis (GenScript Inc.), and the synthetic cDNA was then cloned into the pEXP5NT vector (Invitrogen), and expressed as an N-terminal fusion protein with a 6xHis tag and a TEV cleavage site. The plasmid containing the MPN672 gene was then transformed into the BL21 (DE3) pLysS strain and the recombinant protein production was induced by addition of 0.1 mM IPTG at 37°C for 4 h. The cells were harvested by centrifugation at 2000 × g for 25 min at 4°C. The pellets were resuspended in lysis buffer containing 25 mM Tris/HCl, pH 7.5, 2 mM MgCl2, and 0.4 M NaCl. The cells were lysed by repeated freezing and thawing, and sonication for 2 min in an ice/water bath. After centrifugation at 25,000 × g for 30 min at 4°C, the supernatant was used to purify the recombinant protein by metal affinity chromatography on a Ni-Sepharose (GE Healthcare) resin column, and the Mpn HPRT was eluted with 0.4 M imidazole in lysis buffer. The eluted fractions were analyzed by 12% SDS-PAGE and those containing purified enzyme were pooled and passed through a PD-10 column (GE Healthcare) for desalting and buffer exchange. The final enzyme preparation was in a buffer containing 10 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 20% glycerol, and stored in aliquots at −70°C. Protein concentration was determined by Bio-Rad protein assay using bovine serum albumin (BSA) as a standard.
The HPRT assay was performed by using the DE-81 filter paper assay with tritium labeled hypoxanthine ([3H]-Hx) or guanine ([3H]-Gua) as substrates, essentially as previously described . Briefly, the reaction mixture contained 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 15 mM NaF, 1 mM PRPP, and various concentrations of [3H]-Hx or [3H]-Gua in a total of 50 μl. The reaction was initiated by addition of the enzyme, and at 0, 5, 10, and 15 min intervals, 10 μl reaction mixture was withdrawn and spotted onto the DE81 filter paper and dried. The unreacted substrate was washed and the products were eluted and counted in a liquid scintillation counter. With [3H]-Gua as substrate the reaction (in a total of 25 μl) was initiated by addition of the enzyme (10 μl), incubated at 37°C for 2 min, stopped by addition of 1 M HCl (10 μl), and placed immediately on ice. After neutralization, 15 μl of the mixture was spotted onto the DE81-filter paper. The filters were then washed, and the products were eluted and counted by liquid scintillation.
IC50 values for purine analogs were determined for both Mpn HPRT and human HPRT using fixed concentrations of [3H]-Hx (10 μM) or [3H]-Gua (10 μM) and variable concentrations of the inhibitors.
Thymidine kinase assay was performed using tritium labelled thymidine ([3H]-dT) as substrate and various concentrations of the inhibitors essentially as previously described  to determine the IC50 values of TFT and 5FdU.
Kinetic parameters for TFT were determined by using the phosphoryl transfer assays as previously described . Briefly, each reaction was performed in a total volume of 20 μl containing 50 mM Tris/HCl, pH 7.5, 0.5 mg/ml BSA, 5 mM DTT, 2 mM MgCl2, 15 mM NaF, variable concentrations of TFT, 0.1 mM [γ-32P]-ATP, and 50 ng purified enzyme at 37°C for 20 min, and stopped by heating at 70°C for 2 min. After brief centrifugation, 1 μl supernatant was spotted onto a TLC plate (PEI-cellulose, Merck) and dried. The TLC plates were developed in isobutyric acid/ammonia/H2O (66:1:33). The reaction products were visualized and quantified by phosphoimaging analysis (Quantity One, Bio-Rad).
The data were analysed by unpaired student’s t-test (two tailed) using GraphPad Prism 5 software. P < 0.05 is considered as significant.
This work was supported by a grant from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning. We thank Professor Pär Nordlund, Karolinska Institute, Stockholm, for providing the nucleoside and nucleobase analogs.
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