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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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Influence of T7 DNA primase concentration on reaction yield and product profile. Standard reaction conditions were used as given in the legend of Figure 1 except for T7 DNA primase concentration which was varied between 1 and 8 μM corresponding to the presence of 1000 and 125 pmol of cap analog substrate (7MeGpppA) per pmol T7 DNA primase. DNA template was dAC3 (CCCCGGGTCT25). Reactions were incubated for 21 h and analyzed by HPLC as described in Material and Methods. Yields in percentage conversion of cap analog into each single product (upper panel) and pmol product per pmol enzyme (lower panel) were determined as detailed in Material and Methods. Total product represents the sum of conversion into GpppAC3, GpppAC4 and GpppAC5. Amounts of GpppAC and GpppAC2 products were negligible under the tested reaction conditions.
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Figure 3: Influence of T7 DNA primase concentration on reaction yield and product profile. Standard reaction conditions were used as given in the legend of Figure 1 except for T7 DNA primase concentration which was varied between 1 and 8 μM corresponding to the presence of 1000 and 125 pmol of cap analog substrate (7MeGpppA) per pmol T7 DNA primase. DNA template was dAC3 (CCCCGGGTCT25). Reactions were incubated for 21 h and analyzed by HPLC as described in Material and Methods. Yields in percentage conversion of cap analog into each single product (upper panel) and pmol product per pmol enzyme (lower panel) were determined as detailed in Material and Methods. Total product represents the sum of conversion into GpppAC3, GpppAC4 and GpppAC5. Amounts of GpppAC and GpppAC2 products were negligible under the tested reaction conditions.

Mentions: Enzyme concentration was varied between 1 and 8 μM. As shown in Figure 3, the overall yield and the length of formed products increase with enzyme concentration within the tested range (upper panel). Nevertheless, the efficiency of the enzymatic reaction decreases from 867.5 pmol per pmol enzyme at 1 μM to 125.0 pmol per pmol enzyme at 8 μM enzyme concentration (lower panel). In conclusion, 4 µM T7 DNA primase was kept as enzyme concentration for oligonucleotide production on a preparative scale and further optimization studies.Figure 3.


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)

Influence of T7 DNA primase concentration on reaction yield and product profile. Standard reaction conditions were used as given in the legend of Figure 1 except for T7 DNA primase concentration which was varied between 1 and 8 μM corresponding to the presence of 1000 and 125 pmol of cap analog substrate (7MeGpppA) per pmol T7 DNA primase. DNA template was dAC3 (CCCCGGGTCT25). Reactions were incubated for 21 h and analyzed by HPLC as described in Material and Methods. Yields in percentage conversion of cap analog into each single product (upper panel) and pmol product per pmol enzyme (lower panel) were determined as detailed in Material and Methods. Total product represents the sum of conversion into GpppAC3, GpppAC4 and GpppAC5. Amounts of GpppAC and GpppAC2 products were negligible under the tested reaction conditions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851634&req=5

Figure 3: Influence of T7 DNA primase concentration on reaction yield and product profile. Standard reaction conditions were used as given in the legend of Figure 1 except for T7 DNA primase concentration which was varied between 1 and 8 μM corresponding to the presence of 1000 and 125 pmol of cap analog substrate (7MeGpppA) per pmol T7 DNA primase. DNA template was dAC3 (CCCCGGGTCT25). Reactions were incubated for 21 h and analyzed by HPLC as described in Material and Methods. Yields in percentage conversion of cap analog into each single product (upper panel) and pmol product per pmol enzyme (lower panel) were determined as detailed in Material and Methods. Total product represents the sum of conversion into GpppAC3, GpppAC4 and GpppAC5. Amounts of GpppAC and GpppAC2 products were negligible under the tested reaction conditions.
Mentions: Enzyme concentration was varied between 1 and 8 μM. As shown in Figure 3, the overall yield and the length of formed products increase with enzyme concentration within the tested range (upper panel). Nevertheless, the efficiency of the enzymatic reaction decreases from 867.5 pmol per pmol enzyme at 1 μM to 125.0 pmol per pmol enzyme at 8 μM enzyme concentration (lower panel). In conclusion, 4 µM T7 DNA primase was kept as enzyme concentration for oligonucleotide production on a preparative scale and further optimization studies.Figure 3.

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