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High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions.

Peyrane F, Selisko B, Decroly E, Vasseur JJ, Benarroch D, Canard B, Alvarez K - Nucleic Acids Res. (2007)

Bottom Line: Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times.Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog.Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, AFMB-CNRS-ESIL, Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France.

ABSTRACT
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

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Methyltransferase reactions using GpppAC3 as substrate. (A): HPLC profile of the NS5MTaseDV reaction mixture using GpppAC3 as substrate. Reaction conditions are given in Material and Methods. The crude enzymatic mixture was analyzed without sample treatment. The first section (in gray) indicates the removal of proteic material and remaining AdoMet by on-line cleaning on the pre-column (see Material and Methods). The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min reaching 30% after 45 min. (B): HPLC profile (lower chromatogram) of nucleoside product mixture after enzymatic digestion of the product generated by methylation of GpppAC3 by NS5MTaseDV (see panel A at 35.4 min). Enzymatic digestion was done using a mix of nucleotide pyrophosphatase, phosphodiesterase I and calf intestine phosphatase. The upper chromatogram shows a mixture of standard compounds. No on-line cleaning was used. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min and to 30% after 45 min. (C): HPLC profile of the human mRNA cap N7MTase reaction mixture using GpppAC3 as substrate (lower chromatogram) in comparison to 7MeGpppAC3 being used as a control substrate (upper chromatogram). Reaction conditions are given in Material and Methods. The crude enzymatic mixtures were analyzed without sample treatment as described in the legend of panel A.
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Figure 4: Methyltransferase reactions using GpppAC3 as substrate. (A): HPLC profile of the NS5MTaseDV reaction mixture using GpppAC3 as substrate. Reaction conditions are given in Material and Methods. The crude enzymatic mixture was analyzed without sample treatment. The first section (in gray) indicates the removal of proteic material and remaining AdoMet by on-line cleaning on the pre-column (see Material and Methods). The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min reaching 30% after 45 min. (B): HPLC profile (lower chromatogram) of nucleoside product mixture after enzymatic digestion of the product generated by methylation of GpppAC3 by NS5MTaseDV (see panel A at 35.4 min). Enzymatic digestion was done using a mix of nucleotide pyrophosphatase, phosphodiesterase I and calf intestine phosphatase. The upper chromatogram shows a mixture of standard compounds. No on-line cleaning was used. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min and to 30% after 45 min. (C): HPLC profile of the human mRNA cap N7MTase reaction mixture using GpppAC3 as substrate (lower chromatogram) in comparison to 7MeGpppAC3 being used as a control substrate (upper chromatogram). Reaction conditions are given in Material and Methods. The crude enzymatic mixtures were analyzed without sample treatment as described in the legend of panel A.

Mentions: The purified capped RNA oligonucleotides were tested as substrates of Dengue 2′OMTase, expressed as recombinant N-terminal domain of protein NS5 (NS5MTaseDV) in E. coli (6). In contrast to the usual set-up of methyltransfer assays (15), we used non-radioactive AdoMet and RNA substrate. The reaction mixture was analyzed by reverse-phase HPLC including an on-line cleaning procedure without any additional sample treatment. Figure 4A shows the resulting profile after a 30-min reaction using GpppAC3 as substrate. The retention time of substrate GpppAC3 was 30.6 min. An additional peak was observed at 35.4 min. Its molecular mass was determined by mass spectrometry as being 1702.44 (m/z, [M−H]−) compared to 1688.24 (m/z, [M−H]−, see Table 3) of GpppAC3, thus one methyl group was transferred. In order to identify the receiving position, we digested the product at 35.4 min by a mix of nucleotide pyrophosphatase, phosphodiesterase I and calf intestine phosphatase rendering the corresponding nucleosides. The resulting mixture was analyzed by HPLC (Figure 4B, lower chromatogram). The comparison with standard compounds (upper chromatogram), which was verified by co-injection (not shown), allowed the identification of 2′O position of the adenosine as the methylated position. Thus, in accordance with earlier results using non-purified substrates (6), we found that the product of methyltransfer by NS5MTaseDV using substrate GpppAC3 corresponds to GpppA2′OMeC3. Note that the important delay in elution of GpppA2′OMeC3 in comparison to substrate GpppAC3 is caused by an increase in hydrophobicity due to the methylation of the 2′OH group combined with the use of a very shallow gradient (see figure legend). In order to see if methylation could also be achieved at the guanine-N7 position of GpppAC3, we used recombinant human mRNA cap N7MTase (33,34). The HPLC analysis of the reaction products (Figure 4C, lower chromatogram) in comparison to a control reaction using 7MeGpppAC3 (upper chromatogram) shows that GpppAC3 is indeed efficiently methylated at the guanine-N7 position. Note that in this case, a positive charge is generated upon methylation leading to a shorter elution time of the product (29.3 min) in comparison to the non-methylated substrate (30.7 min). Thus both methyltransfer reactions were readily detectable by our HPLC separation method. They can be easily analyzed in a quantitative way in terms of picomoles methyl group transferred by one picomole enzyme.Figure 4.


High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions.

Peyrane F, Selisko B, Decroly E, Vasseur JJ, Benarroch D, Canard B, Alvarez K - Nucleic Acids Res. (2007)

Methyltransferase reactions using GpppAC3 as substrate. (A): HPLC profile of the NS5MTaseDV reaction mixture using GpppAC3 as substrate. Reaction conditions are given in Material and Methods. The crude enzymatic mixture was analyzed without sample treatment. The first section (in gray) indicates the removal of proteic material and remaining AdoMet by on-line cleaning on the pre-column (see Material and Methods). The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min reaching 30% after 45 min. (B): HPLC profile (lower chromatogram) of nucleoside product mixture after enzymatic digestion of the product generated by methylation of GpppAC3 by NS5MTaseDV (see panel A at 35.4 min). Enzymatic digestion was done using a mix of nucleotide pyrophosphatase, phosphodiesterase I and calf intestine phosphatase. The upper chromatogram shows a mixture of standard compounds. No on-line cleaning was used. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min and to 30% after 45 min. (C): HPLC profile of the human mRNA cap N7MTase reaction mixture using GpppAC3 as substrate (lower chromatogram) in comparison to 7MeGpppAC3 being used as a control substrate (upper chromatogram). Reaction conditions are given in Material and Methods. The crude enzymatic mixtures were analyzed without sample treatment as described in the legend of panel A.
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Related In: Results  -  Collection

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Figure 4: Methyltransferase reactions using GpppAC3 as substrate. (A): HPLC profile of the NS5MTaseDV reaction mixture using GpppAC3 as substrate. Reaction conditions are given in Material and Methods. The crude enzymatic mixture was analyzed without sample treatment. The first section (in gray) indicates the removal of proteic material and remaining AdoMet by on-line cleaning on the pre-column (see Material and Methods). The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min reaching 30% after 45 min. (B): HPLC profile (lower chromatogram) of nucleoside product mixture after enzymatic digestion of the product generated by methylation of GpppAC3 by NS5MTaseDV (see panel A at 35.4 min). Enzymatic digestion was done using a mix of nucleotide pyrophosphatase, phosphodiesterase I and calf intestine phosphatase. The upper chromatogram shows a mixture of standard compounds. No on-line cleaning was used. The gradient started after 5 min at 100% eluent A with an increase to 10% eluent B after 25 min and to 30% after 45 min. (C): HPLC profile of the human mRNA cap N7MTase reaction mixture using GpppAC3 as substrate (lower chromatogram) in comparison to 7MeGpppAC3 being used as a control substrate (upper chromatogram). Reaction conditions are given in Material and Methods. The crude enzymatic mixtures were analyzed without sample treatment as described in the legend of panel A.
Mentions: The purified capped RNA oligonucleotides were tested as substrates of Dengue 2′OMTase, expressed as recombinant N-terminal domain of protein NS5 (NS5MTaseDV) in E. coli (6). In contrast to the usual set-up of methyltransfer assays (15), we used non-radioactive AdoMet and RNA substrate. The reaction mixture was analyzed by reverse-phase HPLC including an on-line cleaning procedure without any additional sample treatment. Figure 4A shows the resulting profile after a 30-min reaction using GpppAC3 as substrate. The retention time of substrate GpppAC3 was 30.6 min. An additional peak was observed at 35.4 min. Its molecular mass was determined by mass spectrometry as being 1702.44 (m/z, [M−H]−) compared to 1688.24 (m/z, [M−H]−, see Table 3) of GpppAC3, thus one methyl group was transferred. In order to identify the receiving position, we digested the product at 35.4 min by a mix of nucleotide pyrophosphatase, phosphodiesterase I and calf intestine phosphatase rendering the corresponding nucleosides. The resulting mixture was analyzed by HPLC (Figure 4B, lower chromatogram). The comparison with standard compounds (upper chromatogram), which was verified by co-injection (not shown), allowed the identification of 2′O position of the adenosine as the methylated position. Thus, in accordance with earlier results using non-purified substrates (6), we found that the product of methyltransfer by NS5MTaseDV using substrate GpppAC3 corresponds to GpppA2′OMeC3. Note that the important delay in elution of GpppA2′OMeC3 in comparison to substrate GpppAC3 is caused by an increase in hydrophobicity due to the methylation of the 2′OH group combined with the use of a very shallow gradient (see figure legend). In order to see if methylation could also be achieved at the guanine-N7 position of GpppAC3, we used recombinant human mRNA cap N7MTase (33,34). The HPLC analysis of the reaction products (Figure 4C, lower chromatogram) in comparison to a control reaction using 7MeGpppAC3 (upper chromatogram) shows that GpppAC3 is indeed efficiently methylated at the guanine-N7 position. Note that in this case, a positive charge is generated upon methylation leading to a shorter elution time of the product (29.3 min) in comparison to the non-methylated substrate (30.7 min). Thus both methyltransfer reactions were readily detectable by our HPLC separation method. They can be easily analyzed in a quantitative way in terms of picomoles methyl group transferred by one picomole enzyme.Figure 4.

Bottom Line: Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times.Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog.Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, AFMB-CNRS-ESIL, Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France.

ABSTRACT
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

Show MeSH
Related in: MedlinePlus