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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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Time course of capped RNA synthesis by T7 DNA primase on DNA template dAC3 (CCCCGGGTCT25). Reaction conditions are given in the legend of Figure 1. Samples were taken at the given time points and analyzed by HPLC. The determination of yields (pmol product per pmol enzyme) is detailed in Material and Methods. (A) global analysis of substrate depletion (▪ GpppA, • 7MeGpppA) and product generation (□ GpppACn, ◯ 7MeGpppACn) with time, (B) formation of individual products in the GpppA-capped RNA product mixture (• GpppAC, ▪ GpppAC2, ♦ GpppAC3, □ GpppAC4, ×GpppAC5).
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Figure 2: Time course of capped RNA synthesis by T7 DNA primase on DNA template dAC3 (CCCCGGGTCT25). Reaction conditions are given in the legend of Figure 1. Samples were taken at the given time points and analyzed by HPLC. The determination of yields (pmol product per pmol enzyme) is detailed in Material and Methods. (A) global analysis of substrate depletion (▪ GpppA, • 7MeGpppA) and product generation (□ GpppACn, ◯ 7MeGpppACn) with time, (B) formation of individual products in the GpppA-capped RNA product mixture (• GpppAC, ▪ GpppAC2, ♦ GpppAC3, □ GpppAC4, ×GpppAC5).

Mentions: Subsequently, we followed product formation over time in order to reach optimal substrate conversion (Figure 2). Figure 2A shows the overall yield when GpppA or 7MeGpppA were used as cap analogs. T7 DNA primase incorporates both to same extent. Reactions were completed to 96% after 21 h. Figure 2B shows accumulation of individual products GpppAC to GpppAC5 of one of the reactions using GpppA as cap analog. GpppAC was produced during the early phase of reaction and completely converted into longer products after 16 h. GpppAC2 was equally only present during the early phase of the reaction but to very low extent. After 1 h, GpppAC3 was the main product and remained so throughout the reaction. GpppAC4 appeared almost immediately and its yield grew linearly, whereas GpppAC5 appeared after 15 h. Thus leaving the reaction longer opens the possibility to produce higher amounts of longer substrates (see below). The product profiles were similar when 7MeGpppA was used as cap analog (not shown).Figure 2.


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)

Time course of capped RNA synthesis by T7 DNA primase on DNA template dAC3 (CCCCGGGTCT25). Reaction conditions are given in the legend of Figure 1. Samples were taken at the given time points and analyzed by HPLC. The determination of yields (pmol product per pmol enzyme) is detailed in Material and Methods. (A) global analysis of substrate depletion (▪ GpppA, • 7MeGpppA) and product generation (□ GpppACn, ◯ 7MeGpppACn) with time, (B) formation of individual products in the GpppA-capped RNA product mixture (• GpppAC, ▪ GpppAC2, ♦ GpppAC3, □ GpppAC4, ×GpppAC5).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 2: Time course of capped RNA synthesis by T7 DNA primase on DNA template dAC3 (CCCCGGGTCT25). Reaction conditions are given in the legend of Figure 1. Samples were taken at the given time points and analyzed by HPLC. The determination of yields (pmol product per pmol enzyme) is detailed in Material and Methods. (A) global analysis of substrate depletion (▪ GpppA, • 7MeGpppA) and product generation (□ GpppACn, ◯ 7MeGpppACn) with time, (B) formation of individual products in the GpppA-capped RNA product mixture (• GpppAC, ▪ GpppAC2, ♦ GpppAC3, □ GpppAC4, ×GpppAC5).
Mentions: Subsequently, we followed product formation over time in order to reach optimal substrate conversion (Figure 2). Figure 2A shows the overall yield when GpppA or 7MeGpppA were used as cap analogs. T7 DNA primase incorporates both to same extent. Reactions were completed to 96% after 21 h. Figure 2B shows accumulation of individual products GpppAC to GpppAC5 of one of the reactions using GpppA as cap analog. GpppAC was produced during the early phase of reaction and completely converted into longer products after 16 h. GpppAC2 was equally only present during the early phase of the reaction but to very low extent. After 1 h, GpppAC3 was the main product and remained so throughout the reaction. GpppAC4 appeared almost immediately and its yield grew linearly, whereas GpppAC5 appeared after 15 h. Thus leaving the reaction longer opens the possibility to produce higher amounts of longer substrates (see below). The product profiles were similar when 7MeGpppA was used as cap analog (not shown).Figure 2.

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