Enzymatic synthesis of DNA employing pyrophosphate-linked dinucleotide substrates
© Song et al; licensee Chemistry Central Ltd. 2011
Received: 9 August 2011
Accepted: 4 October 2011
Published: 4 October 2011
One of the remaining questions in the understanding of the origin of Nature's information system is the way the first nucleic acids have been synthesized. This could have been realized using nucleoside triphosphates or imidazolides of nucleoside monophosphates as building blocks. Alternatively, dinucleoside pyrophosphates could have been used for this purpose. The advantage of using building blocks, composed of pyrophosphate-linked dinucleotides, could be that exponential growth of initial information (dinucleotides) without product inhibition might become possible.
Herein, we demonstrate that dinucleoside pyrophosphates are able to act as substrate for HIV-1 RT and several thermostable DNA polymerases. In single incorporation assay, compound dAppdA was able to give a 100% conversion to the (P+1) strand by Therminator DNA polymerase and at a substrate concentration above 100 μM. Full-length elongation was obtained in a chain elongation experiment, with over 95% yield of (P+7) product by Taq and Vent (exo-) DNA polymerase. Interestingly, using heterodimer dAppdT addition of either nucleotide component of the dinucleotide substrate into the DNA chain can occur, which is defined by the template program.
This study shows that dinucleoside pyrophosphates can be considered as a new type of substrate for polymerases in the template-directed DNA synthesis. Using heterodimers as substrate, theoretically, it is possible to synthesize DNA enzymatically using two building blocks (dAppdT and dGppdC) instead of four. Given the poor Km value for the nucleotide incorporation, evolution of polymerases will become necessary to make this process of practical use.
Dinucleotides are chemically easily available, demonstrated by the observation that the reaction of ImpA with glycolic acid in the presence of divalent metal ions may lead to AppA formation . We also observed that L-Asp-dTMP may dimerize in solution to give dTppdT (unpublished results). The use of pyrophosphate linked nucleotides as building blocks for nucleic acid synthesis has not been investigated so far.
Overview of the dinucleoside pyrophosphates used in this study.
31P NMR, ppm
Results and Discussion
Synthesis of dinucleoside diphosphates
Single nucleotide incorporation by HIV-1 RT
Overview of primer and template sequences used in the DNA polymerase reaction[a]
Since HIV-1 RT is an error-prone polymerase with high mutation rate, it exhibits flexibility and high tolerance towards chemically modified nucleotide substrate [18, 19]. Therefore, we firstly select HIV-1 RT to perform the single nucleotide incorporation experiments in a primer-template assay. The building blocks were incubated at 37°C, with the appropriate primer-template complex and 0.025 U/μL of enzyme, samples were taken after 10, 20, 30, 60, and 120 min, and analyzed by polyacrylamide gel electrophoresis.
Single nucleotide incorporation by other DNA polymerases
After obtaining the single nucleotide incorporation results of compound I-VII with HIV-1 RT, we tested the single incorporation properties of compound I-VII with a selection of thermostable DNA polymerases.
Compound II, which shows similar substrate property as compound I using HIV-1 RT as polymerase, can achieve 70% conversion to (P+1) strand at 500 μM substrate concentration in 60 min and 100% conversion at 1 mM by Therminator DNA polymerase (Figure 4d). However, compound II was not a substrate for Taq and Vent (exo-) DNA polymerase, as no (P+1) product formation was observed at the concentrations ranging from 100 μM to 1 mM (data not shown).
Single incorporation results of compound III-VII by Taq, Vent (exo-) and Therminator DNA polymerase.
Single incorporation result, %
Vent (exo - )
Therminator is a mutant variant of the 9°N exo- polymerase (Thermococcus species 9°N-7), in which the Ala 485 residue has been replaced with Leu residue. This enzyme demonstrated effective ability of recognition and incorporation of a number of modified nucleotides bearing unnatural nucleobase and sugar moieties . Using compound III-VI as substrates, Therminator polymerase was able to insert deoxyadenylate, deoxythymidylate and deoxyguanylate residue into the DNA primer-template complex (Table 3) depending on the template program, however, it fails to incorporated the deoxycytidylate residue into the growing primer strand as observed for HIV-1 RT. The observed diversity in incorporation selectivity for various polymerases (HIV-1 RT, Taq, Vent (exo-)) indicates the differences in tolerance of the active site of polymerase to the triphosphate modifications.
Primer extension experiments
So far, compound I show the best results in single nucleotide incorporation experiments towards various polymerases. We decided to further investigate the chain elongation property of compound I by HIV-1 RT, Taq, Vent (exo-) and Therminator DNA polymerase, respectively. In this experiment a template overhang with seven thymidines and flanked by four non-thymidine nucleotides at the 3'-end was used. The building block was incubated at the appropriate temperature in a concentration of 500 μM, with primer P1 and template T3 complex and appropriate concentration of enzyme, samples were taken after 15, 30, 60, 90, and 120 min, and analyzed by polyacrylamide gel electrophoresis.
The kinetic parameters for incorporation of the natural nucleotide (dAMP) and compound I into P1T1 by HIV-1 RT.
35 ± 2
0.3 ± 0.1
116 × 10-3
37 ± 5
447 ± 117
0.08 × 10-3
As seen in Table 4, compound I has the similar Vmax value as the natural substrate dATP. However, a large increase in the KM value for the incorporation of compound I was observed, this implies a decrease in the enzymatic affinity for dinucleoside pyrophosphate compared to dATP. Thus, the measured ratio (Vmax/KM) toward HIV-1 RT is 1400-fold decreased compared to that of dATP. It can be assumed that although compound I was rather efficient as a substrate into a growing DNA strand, the enzyme still prefer the natural dATP as substrate when giving a choice.
In an earlier enzymatic incorporation study when thermostable DNA polymerases were used, it was observed that the chemical stability of dNTP analogues at elevated temperature (70°C) influenced the efficiency of incorporation . In this study we have determined the chemical stability of dAppdA, dGppdG, dCppdC, dTppdT and dGppdC analogues in the pH range 2-13 at room temperature (25°C) and 70°C using NMR spectroscopy. All the analogues are found to be stable (no degradation product identified after 3 days) in pH range 7-12 at 25°C and 70°C. Generally dNppdN analogues are very stable, and they degrade to nucleotides and nucleosides slowly above pH 12 at 70°C and degrade rapidly at less than pH 3, 70°C. In comparison the N-glycosidic bond of dAppdA, dGppdG, dCppdC analogues is weaker than the P-O-P linkage in acidic medium, since degradation of glycosidic bond was observed in pH range 4-6.5 at 70°C.
Relevance to prebiotic chemistry
The process may be run without waste of material as the nucleotide leaving group is reusable (condensation of pX and pY to the XppY reagent in the presence of an activating agent).
It is obvious that for every reaction there will be a competition between phosphodiester formation between the nucleotides away from the template (Scheme 2a) and between the nucleotides recognized by the template (Scheme 2b).
In the latter case (Scheme 2b, Scheme 3b), we end up in the classical trap of product inhibition. In the first case (Scheme 2a), exponential growth of information without product inhibition may become possible.
Using 4 nucleotides, however, in total 10 possible pyrophosphate linked dinucleotides can be formed from which 4 can be recognized by a homodimer, which makes it unlikely that a process including all 4 nucleotides would have an advantage for prebiotic synthesis of nucleic acids (when compared with 4 nucleoside triphosphates).
Once a diverse set of short oligomers is formed in this way, they can be assembled via Watson-Crick base pairing and grow to a larger self-complementary information system (Scheme 4).
However, given the high stability of the pyrophosphate linker, it will not be easy to find an appropriate catalyst for these reactions.
The experimental set-up in this monograph has demonstrated that initial DNA dependent DNA polymerase activity can be ascribed to HIV-RT and several thermostable DNA polymerases using dinucleoside pyrophosphates as substrates. This means that, when using such heterodimers for DNA synthesis (for example dAppdT and dGppdC), theoretically, it is possible to synthesize DNA enzymatically using two building blocks instead of four. These initial experiments have been carried out in the light of trying to find model compounds that could have been used in a primitive life form to build up an information system while still having the capacity for exponential growth of this information.
The general principle that has been used by Nature for protein synthesis, i.e., to incorporate information in leaving groups, may as well have been used by Nature to select its most abundant information system itself. Future experiments are aimed to find the appropriate X (Figure 9) together with the optimal reaction circumstances to realize metabolic cycles as depicted in Figures 7 and 8.
For all reactions, chemicals of analytical or synthetic grade were obtained from commercial sources and were used without purification. Technical solvents were obtained from Brenntag (Deerlijk, Belgium). Analytical thin Layer Chromatography was performed on Alugram® silica gel UV254 mesh 60, 0.20 mm (Macherey-Nagel). NMR Spectra were recorded on a Brucker Avance™ II 300 MHz or 500 MHz NMR spectrometer. Chemical shifts are expressed as δ units (part per million) down field from TMS (tetramethyl silane) for 1H and 13C. 31P NMR chemical shifts are referenced to an external 85% H3PO4 standard (δ = 0.00 ppm). Exact mass spectra were obtained with a quadrupol/orthogonal-acceleration time-of-flight tandem mass spectrometer (Q-Tof 2, Micromass) equipped with a standard electrospray ionization (ESI) source. HPLC was performed on Waters 1525-2487 system using C18 column by a gradient elution of acetonitrile and 50 mM triethylammonium bicarbonate (TEAB) buffer.
Synthesis of deoxyadenosine-5'-phosphoroimidazole
General Procedure (I): Deoxyadeosine-5'-monophosphate (100 mg, 0.30 mmoles) was dissolved in dry DMSO (2 mL), pulverized triphenylphosphine (327 mg, 0.96 mmoles), 2,2-dipyridyl disulfide (211 mg, 0.96 mmoles) and imidazole (327 mg, 4.8 mmoles) were added to the solution, the resulting yellow solution was stirred at room temperature for 50 min, after completion the mixture was poured into 0.1 M sodium iodide solution in cold acetone (30 mL). The white precipitate was collected by centrifugation and washed several times with cold fresh acetone. The product was dried in desicator for 1 h.; the white solid was stored at -20°C (99 mg, yield 87%). 1H NMR (500 MHz, D2O): δ= 8.07 (s, 1H, H8), 7.92 (s, 1H, H2), 6.20 (t, 1H, J = 6.2 Hz, H1'), 4.55 (m, 1H, H3'), 4.13 (m, 3H, H4'+H5'), 2.50 (m, 1H, H2'a), 2.43 (m, 1H, H2'b); 13C NMR (125 MHz, D2O): δ = 155.1, 152.6, 148.4, 140.1, 118.4, 86.5, 84.4, 71.2, 66.0, 40.0; 31P NMR (121 MHz, D2O): δ = -11.23; HRMS: [M-H]- calculated for C20H25N10O11P2 643.1185, found: 643.1183.
Synthesis of P1, P2-bis(2'-deoxyadenosin-5'-yl) Pyrophosphate (dAppdA)
General Procedure (II): A solution of 2'-deoxyadeonosine-5'-monophosphate (100 mg, 0.30 mmoles), 2'-deoxyadeonosine-5'-phosphorimidazolide (114 mg, 0.30 mmoles), zinc chloride (35 mg, 0.30 mmoles) in 0.2 M N-ethylmorpholine buffer (3 mL, pH 7.5) was stirred at room temperature under argon for 2 days. The reaction was monitored by TLC (i-PrOH/NH3/H2O 7:1:2) and 31P NMR, quenched the reaction with 0.25 M ethylenediaminetetraacetic acid (EDTA) in order to breakdown the nucleotide-metal complex, then the mixture was lyophilized affording a white solid. The product was isolated by HPLC purification on a C18 column, running with a gradient of CH3CN in 50 mM triethylammonium (TEAB) buffer. Yield 28% (from HPLC purification profile). 1H NMR (500 MHz, D2O): δ = 8.02 (s, 2H, H8), 7.90 (s, 2H, H2), 6.16 (t, 2H, J = 5.7 Hz, H1'), 4.49 (m, 2H, H3'), 4.07 (m, 4H, H5'), 4.02 (m, 2H, H4'), 2.46 (m, 2H, H2'a), 2.40 (m, 2H, H2'b); 13C NMR (125 MHz, D2O): δ = 155.1, 152.3, 148.4, 140.1, 118.4, 86.1, 84.1, 71.2, 66.0, 40.0; 31P NMR (121 MHz, D2O): δ = -11.27; HRMS: [M-H]- calculated for C20H25N10O11P2 643.1185, found: 643.1183.
Synthesis of P1, P2-bis(2'-deoxythymidin-5'-yl) Pyrophosphate (dTppdT)
The general procedure (I) was applied using thymidine-5'-monophosphate (300 mg, 0.93 mmoles), triphenylphosphine (1.01 g, 2.98 mmoles), 2,2-dipyridyl disulfide (655 mg, 2,98 mmoles) and imidazole (1.02 g, 14.9 mmoles). After reaction the obtained product was applied to general procedure (II) using thymidine-5'-monophosphate (100 mg, 0.31 mmoles), thymidine-5'-phosphorimidazolide (115 mg, 0.31 mmoles) and zinc chloride (36 mg, 0.31 mmoles). After purification obtained white solid product (yield 35% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 7.71 (s, 2H, H6), 6.29 (t, 2H, J = 7.0 Hz, H1'), 4.56-4.61 (m, 2H, H3'), 4.16-4.17 (m, 2H, H4'), 4.12-4.13 (m, 4H, H5'), 2.32-2.36 (m, 4H, H2'), 1.91 (s, 6H, CH3); 13C NMR (125 MHz, D2O): δ = 166.1, 151.3, 137.1, 111.4, 85.0, 84.9, 70.5, 65.0, 38.3, 11.4; 31P NMR (121 MHz, D2O): δ = -11.62; HRMS: [M-H]- calculated for C20H28N4O15P2 625.0953, found: 625.0962
Synthesis of P1-(2'-deoxyadenosin-5'-yl) P2-(2'-deoxythymidin-5'-yl) Pyrophosphate (dAppdT)
The general procedure (II) was applied using 2'-deoxyadeonosine- 5'-phosphorimidazolide (100 mg, 0.26 mmoles) and thymidine-5'-monophosphate (85 mg, 0.26 mmoles) and zinc chloride (30 mg, 0.26 mmoles). After purification obtained white solid product (yield 18% (from HPLC purification profile)) 1H NMR (500 MHz, D2O): δ = 8.36 (s, 1H, H8-A), 8.10 (s, 1H, H2-A), 7.31 (s, 1H, H6-T), 6.39 (t, 1H, J = 6.0 Hz, H1'-A), 6.15 (t, 1H, J = 6.0 Hz, H1'-T), 4.63-4.65 (m, 1H, H3'-A), 4.40-4.42 (m, 1H, H3'-T), 4.16-4.18 (m, 1H, H4'-A), 4.07-4.00 (m, 5H, H4'-T, H5'-A, H5'-T), 2.71-2.75 (m, 1H, H2'a-A), 2.46-2.50 (m, 1H, H2'a-T), 2.11-2.15 (m, 2H, H2'b-T), 2.04-2.08 (m, 2H, H2'b-A), 1.67 (s, 3H, T-CH3); 13C NMR (125 MHz, D2O): δ = 181.4, 172.3, 155.3, 152.4, 148.5, 139.7, 136.1, 118.4, 111.4, 85.6, 84.8, 84.6, 83.3, 71.0, 71.1, 65.4, 65.3, 38.9, 38.6, 12.3; 31P NMR (121 MHz, D2O): δ = -11.41; HRMS: [M-H]- calculated for C20H26N7O13P2 634.1069, found: 634.1063.
Synthesis of P1, P2-bis(2'-deoxyguanosin-5'-yl) Pyrophosphate (dGppdG)
The general procedure (I) was applied using using 2'-deoxyguanosine-5'-monophosphate (300 mg, 0.78 mmoles), triphenylphosphine (850 mg, 2.50 mmoles), 2,2-dipyridyl disulfide (549 mg, 2,50 mmoles) and imidazole (850 mg, 12.9 mmoles). After reaction the obtained product was applied to general procedure (II) using 2'-deoxyguanosine-5'-monophosphate (100 mg, 0.26 mmoles), 2'-deoxyguanosine-5'-phosphorimidazolide (102 mg, 0.26 mmoles) and Pd(NO3)2 (60 mg, 0.36 mmoles). After purification obtained white solid product (yield 33% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 7.86 (s, 2H, H6), 6.11 (t, 2H, J = 6.5 Hz, H1'), 4.54-4.56 (m, 2H, H3'), 4.05-4.07 (m, 2H, H4'), 4.03-4.05 (m, 4H, H5'), 2.54-2.63 (m, 2H, H2'a), 2.33-2.36 (m, 2H, H2'b); 13C NMR (125 MHz, D2O): δ = 162.1, 156.3, 150.8, 136.3, 116.3, 85.1, 83.0, 70.8, 65.2, 38.4; 31P NMR (121 MHz, D2O): δ = -11.40; HRMS: [M-H]- calculated for C20H25N10O13P2 675.1083, found: 675.1092.
Synthesis of P1-(2'-deoxyguanosin-5'-yl) P2-(2'-deoxycytidin-5'-yl) Pyrophosphate (dGppdC)
The general procedure (I) was applied using using 2'-deoxyguanosine-5'-monophosphate (300 mg, 0.78 mmoles), triphenylphosphine (850 mg, 2.50 mmoles), 2,2-dipyridyl disulfide (549 mg, 2,50 mmoles) and imidazole (850 mg, 12.9 mmoles). After reaction the obtained product was applied to general procedure (II) using 2'-deoxyguanosine-5'-phosphorimidazolide (100 mg, 0.25 mmoles), 2'-deoxycytidine-5'-monophosphate (82 mg, 0.25 mmoles) and Pd(NO3)2 (58 mg, 0.25 mmoles). After purification obtained white solid product (yield 15% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 8.04 (s, 1H, H8-G), 7.82 (d, 1H, J = 7.5 Hz, H6-C), 6.30 (t, 1H, J = 7.0 Hz, H1'-G), 6.25 (t, 1H, J = 6.5 Hz, H1'-C), 5.97 (d, 1H, J = 7.0 Hz, H5-C), 4.63-4.67 (m, 1H, H3'-G), 4.52-4.54 (m, 1H, H3'-C), 4.23-4.25 (m, 1H, H4'-G), 4.15-4.17 (m, 1H, H4'-C), 4.12-4.15 (m, 4H, H5'-G, H5'-C), 2.77-2.82 (m, 1H, H2'a-G), 2.48-2.50 (m, 1H, H2'a-C), 2.36-2.41 (m, 1H, H2'b-C), 2.18-2.24 (m, 1H, H2'b-G); 13C NMR (125 MHz, D2O): δ = 165.6, 161.5, 156.9, 150.9, 140.9, 136.5, 116.3, 95.9, 85.5, 85.2, 85.0, 83.0, 70.9, 70.2, 65.3, 64.8, 39.2, 38.1; 31P NMR (121 MHz, D2O): δ = -11.40; HRMS: [M-H]- calculated for C19H25N8O13P2 635.1022, found: 635.1099.
Synthesis of P1-(2'-deoxyadenosin-5'-yl) P2-(2'-deoxythymidin-5'-yl) Pyrophosphate (dAppdC)
The general procedure (II) was applied using 2'-deoxyadeonosine-5'-phosphorimidazolide (100 mg, 0.26 mmoles), 2'-deoxycytidine-5'-monophosphate (85 mg, 0.26 mmoles) and zinc chloride (30 mg, 0.26 mmoles). After purification obtained white solid product (yield 18% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 8.38 (s, 1H, H8-A), 8.10 (s, 1H, H2-A), 7.53 (d, 1H, J = 6.0 Hz, H6-C), 6.40 (t, 1H, J = 5.5 Hz, H1'-A), 6.08 (t, 1H, J = 5.5 Hz, H1'-C), 5.68 (d, 1H, J = 6.5 Hz, H5-C), 4.65-4.67 (m, 1H, H3'-A), 4.37-4.39 (m, 1H, H3'-C), 4.20-4.21 (m, 1H, H4'-A), 4.09-4.05 (m, 5H, H5'-A, H5'-C, H4'-C), 2.73-2.78 (m, 1H, H2'a-A), 2.47-2.51 (m, 1H, H2'a-C), 2.22-2.26 (m, 1H, H2'b-C), 1.99-2.03 (m, 1H, H2'b-A); 13C NMR (125 MHz, D2O): δ = 165.5, 157.0, 155.3, 152.4, 148.5, 140.6, 139.6, 118.3, 95.8, 85.7, 85.6, 85.2, 83.4, 71.2, 70.4, 65.5, 65.1, 39.5, 38.8; 31P NMR (121 MHz, D2O): δ = -11.34; HRMS: [M-H]- calculated for C20H25N8O12P2 619.1073, found: 619.1089
Synthesis of P1, P2-bis(2'-deoxycytidin-5'-yl) Pyrophosphate (dCppdC)
The general procedure (I) was applied using 2'-deoxycytidine-5'-monophosphate (300 mg, 0.93 mmoles), triphenylphosphine (1.01 g, 2.98 mmoles), 2,2-dipyridyl disulfide (655 mg, 2,98 mmoles) and imidazole (1.02 g, 14.9 mmoles). After reaction the obtained product was applied to general procedure (II) using 2'-deoxycytidine-5'-monophosphate (100 mg, 0.31 mmoles), 2'-deoxycytidine-5'-phosphorimidazolide (115 mg, 0.31 mmoles) and zinc chloride (36 mg, 0.31 mmoles). After purification obtained white solid product (yield 26% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 7.90 (d, J = 7.6 Hz, 2H, H6), 6.31 (t, 2H, J = 6.8 Hz, H1'), 6.05 (d, 2H, J = 7.5 Hz, H5), 4.52-4.56 (m, 2H, H3'), 4.14-4.18 (m, 6H, H4'+H5'), 2.35-2.41 (m, 2H, H2'a), 2.19-2.28 (m, 2H, H2'b); 13C NMR (125 MHz, D2O): δ = 165.7, 157.2, 141.2, 96.2, 85.5, 85.0, 70.4, 64.9, 39.2; 31P NMR (121 MHz, D2O): δ = -8.84; HRMS: [M-H]- calculated for C18H25N6O13P2 595.0960, found: 595.0954.
Oligodeoxyribonucleotides P1, P2, T1-T6 were purchased from Sigma Genosys. The concentrations were measured with a Varian Cary-300-Bio UV spectrophotometer. The lyophilized oligonucleotides were dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at -20°C. The primer oligonucleotides were 5'-33P-labeled with 5'-[γ33P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to standard procedures. The labelled oligonucleotide was further purified using Illustra TM Microspin TM G-25 columns (GE Healthcare).
DNA polymerase reactions
End-labelled primer was annealed to its template by combining primer and template in a molar ratio of 1:2 and heating the mixture to 70°C for 10 min followed by slow cooling to room temperature over a period of 1.5 h. For the incorporation of compound I-VII, a series of 20 μL-batch reactions were performed with the enzyme (HIV-1 RT, Taq, Vent (exo-), Therminator DNA polymerase). The final mixture contained 125 nM primer template complex, RT buffer (250 mM Tris.HCl, 250 mM KCl, 50 mM MgCl2, 2.5 mM spermidine, 50 mM dithiothreitol (DTT); pH 8.3), appropriate concentration of enzyme, and different concentrations of dinucleoside diphosphates building blocks (1 mM, 500 μM, 200 μM and 100 μM). In the control reaction a 10 μM dATP was used as reference. The mixture was incubated at 37°C or 75°C respectively, and aliquots were quenched after 10, 20, 30, 60 and 120 min. For elongation experiments, the same mixture with appropriate primer/template hybrid was incubated at 37°C or 75°C and aliquots were quenched after 15, 30, 60, 90, 120 min.
Steady-state kinetics of single nucleotide incorporation
The steady-state kinetics of single nucleotide incorporation of compound I and the natural nucleoside triphosphate (dATP) was determined by a gel-based polymerase assay. In all the experiments, the template T1 and the primer P1 were used. The primer and template in a 1:2 molar ratio were hybridised in a buffer containing 20 mM Tris.HCl, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.3 and used in an amount to provide 125 nM concentration of the primer in each 20 μL reaction. A range of building block concentrations between 10 μM and 1 mM for the phosphoramidate and between 0.1 μM and 10 μM for the natural building block was used. The final concentrations of primer-template complex and HIV-1 RT were 125 nM, and 0.025 U/μL, respectively. Reaction mixtures were incubated at 37°C and aliquots were drawn at 6 different time intervals. The reactions were quenched by addition of a buffer containing 80% formamide, 2 mM EDTA and 1X TBE buffer. The analysis of polymerase reaction was performed by polyacrylamide gel electrophoresis (see detailed protocol below). The incorporation rates (V) were calculated based on the percentage of the extended oligonucleotide in the mixture (P+1 band). The kinetic parameters (VMax and KM) were determined by plotting V (nM/min-1) versus substrate concentration (μM) and fitting the data to a non-linear Michaelis-Menten regression using GraphPad Prism Software version 5.0.
All polymerase reaction aliquots (2.5 μL) were quenched by the addition of 10 μL of loading buffer (90% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol and 50 mM ethylenediaminetetraacetic acid). Samples were heated at 85°C for 3 minutes prior to analysis by electrophoresis for 2.5 h at 2000 V on a 30 cm × 40 cm × 0.4 mm 20% (19:1 mono:bis) denaturing gel in the presence of a 100 mM Tris-borate, 2.5 mM EDTA buffer; pH 8.3. Products were visualised by phosphorimaging. The amount of radioactivity in the bands corresponding to the products of enzymatic reactions was determined by using the imaging device Cyclone® and the Optiquant image analysis software (PerkinElmer).
This work was financially supported by a grant from K. U. Leuven (GOA). We would like to thank Prof. Jef Rozenski for providing HRMS, Luc Baudemprez for recording the NMR spectra, and Chantal Biernaux for editorial help.
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