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In vitro selection of a 5'-purine ribonucleotide transferase ribozyme.

Kang TJ, Suga H - Nucleic Acids Res. (2007)

Bottom Line: This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS.Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates.This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan.

ABSTRACT
Here we report in vitro selection of a novel ribozyme that catalyzes the 5'-nucleotidyl transfer reaction forming the 2'-5' phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5'-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3'-->5' mononucleotide extension forming the 2'-5' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

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Product characterizations. (A) Dephosphorylation of the reaction product of M4 and [α-32P]-ATP. Positions where ATP and phosphate (Pi) migrate are shown; AP, alkaline phosphatase; N, AP-non-treated product RNA. (B) DEAE-cellulose TLC analysis of [32P]-labeled nucleotides generated by RNase T2 digestion of the 5′-end-labeled M4 (sample 1), and the reaction product of M4 and [γ-32P]-ATP (sample 2). (C) Construct of trans-acting ribozyme (rM4) and substrate strand (sM4). (D) MALDI-TOF analysis of starting sM4 (upper) and the GMP-αS adduct purified on APM–PAGE (lower); 1, pppGGGAUCGUA [M/z = 3200.923 (calc. 3200)]; 2, SpG(2′)p(5′)GGGAUCGUA [M/z = 3402.989 (calc. 3401); Sp, thiophosphate]. Series of sodium adducts are shown in both spectra as well as dephosphorylated sM4, ppGGGAUCGUA (1′, M/z = 3120.917), in the spectrum for the starting material (upper). (E) The chemistry catalyzed by rM4.
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Figure 4: Product characterizations. (A) Dephosphorylation of the reaction product of M4 and [α-32P]-ATP. Positions where ATP and phosphate (Pi) migrate are shown; AP, alkaline phosphatase; N, AP-non-treated product RNA. (B) DEAE-cellulose TLC analysis of [32P]-labeled nucleotides generated by RNase T2 digestion of the 5′-end-labeled M4 (sample 1), and the reaction product of M4 and [γ-32P]-ATP (sample 2). (C) Construct of trans-acting ribozyme (rM4) and substrate strand (sM4). (D) MALDI-TOF analysis of starting sM4 (upper) and the GMP-αS adduct purified on APM–PAGE (lower); 1, pppGGGAUCGUA [M/z = 3200.923 (calc. 3200)]; 2, SpG(2′)p(5′)GGGAUCGUA [M/z = 3402.989 (calc. 3401); Sp, thiophosphate]. Series of sodium adducts are shown in both spectra as well as dephosphorylated sM4, ppGGGAUCGUA (1′, M/z = 3120.917), in the spectrum for the starting material (upper). (E) The chemistry catalyzed by rM4.

Mentions: Reaction product of M4 with [α-32P]-ATP resulted in radiolabeled RNA as describe above. The resulting RNA was subjected to alkaline phosphatase-mediated dephosphorylation reaction. If M4 catalyzes the capping reaction, the resulting structure that contains a phosphoanhydride bond should be inert toward this enzyme action. However, the transferred [32P]-radiolabel on M4 was readily removed as a phosphate in an alkaline phosphatase-dependent manner, enabling us to rule out the capping reaction from the possible chemistry (Figure 4A).Figure 4.


In vitro selection of a 5'-purine ribonucleotide transferase ribozyme.

Kang TJ, Suga H - Nucleic Acids Res. (2007)

Product characterizations. (A) Dephosphorylation of the reaction product of M4 and [α-32P]-ATP. Positions where ATP and phosphate (Pi) migrate are shown; AP, alkaline phosphatase; N, AP-non-treated product RNA. (B) DEAE-cellulose TLC analysis of [32P]-labeled nucleotides generated by RNase T2 digestion of the 5′-end-labeled M4 (sample 1), and the reaction product of M4 and [γ-32P]-ATP (sample 2). (C) Construct of trans-acting ribozyme (rM4) and substrate strand (sM4). (D) MALDI-TOF analysis of starting sM4 (upper) and the GMP-αS adduct purified on APM–PAGE (lower); 1, pppGGGAUCGUA [M/z = 3200.923 (calc. 3200)]; 2, SpG(2′)p(5′)GGGAUCGUA [M/z = 3402.989 (calc. 3401); Sp, thiophosphate]. Series of sodium adducts are shown in both spectra as well as dephosphorylated sM4, ppGGGAUCGUA (1′, M/z = 3120.917), in the spectrum for the starting material (upper). (E) The chemistry catalyzed by rM4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Product characterizations. (A) Dephosphorylation of the reaction product of M4 and [α-32P]-ATP. Positions where ATP and phosphate (Pi) migrate are shown; AP, alkaline phosphatase; N, AP-non-treated product RNA. (B) DEAE-cellulose TLC analysis of [32P]-labeled nucleotides generated by RNase T2 digestion of the 5′-end-labeled M4 (sample 1), and the reaction product of M4 and [γ-32P]-ATP (sample 2). (C) Construct of trans-acting ribozyme (rM4) and substrate strand (sM4). (D) MALDI-TOF analysis of starting sM4 (upper) and the GMP-αS adduct purified on APM–PAGE (lower); 1, pppGGGAUCGUA [M/z = 3200.923 (calc. 3200)]; 2, SpG(2′)p(5′)GGGAUCGUA [M/z = 3402.989 (calc. 3401); Sp, thiophosphate]. Series of sodium adducts are shown in both spectra as well as dephosphorylated sM4, ppGGGAUCGUA (1′, M/z = 3120.917), in the spectrum for the starting material (upper). (E) The chemistry catalyzed by rM4.
Mentions: Reaction product of M4 with [α-32P]-ATP resulted in radiolabeled RNA as describe above. The resulting RNA was subjected to alkaline phosphatase-mediated dephosphorylation reaction. If M4 catalyzes the capping reaction, the resulting structure that contains a phosphoanhydride bond should be inert toward this enzyme action. However, the transferred [32P]-radiolabel on M4 was readily removed as a phosphate in an alkaline phosphatase-dependent manner, enabling us to rule out the capping reaction from the possible chemistry (Figure 4A).Figure 4.

Bottom Line: This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS.Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates.This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan.

ABSTRACT
Here we report in vitro selection of a novel ribozyme that catalyzes the 5'-nucleotidyl transfer reaction forming the 2'-5' phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-gammaS. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5'-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3'-->5' mononucleotide extension forming the 2'-5' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

Show MeSH