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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.

Show MeSH
Selection of 5′-ppp-dependent cis-thiophosphorylating ribozymes from a random pool. 5′-ppp random RNAs were subjected to the reaction with ATP-γS and the resulting thiophosphorylated RNAs were enriched using the following two methods. In round 1–9, active RNAs were isolated on thiopropyl-activated resin. In round 10–14, active RNAs were modified by biotin-iodoacetoamide and isolated on the streptavidin-agarose resin. It should be noted that the combination of these two procedures turned out to be effective for diminishing the appearance of non-specific aptamers that bind to resins. To knock down the population of 5′-ppp-dependent species in pool 14, the counterselection was performed (right); the 5′-ppp group was removed by alkaline phosphatase (AP) to expose the 5′-OH group in pool 14 RNA.
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Figure 1: Selection of 5′-ppp-dependent cis-thiophosphorylating ribozymes from a random pool. 5′-ppp random RNAs were subjected to the reaction with ATP-γS and the resulting thiophosphorylated RNAs were enriched using the following two methods. In round 1–9, active RNAs were isolated on thiopropyl-activated resin. In round 10–14, active RNAs were modified by biotin-iodoacetoamide and isolated on the streptavidin-agarose resin. It should be noted that the combination of these two procedures turned out to be effective for diminishing the appearance of non-specific aptamers that bind to resins. To knock down the population of 5′-ppp-dependent species in pool 14, the counterselection was performed (right); the 5′-ppp group was removed by alkaline phosphatase (AP) to expose the 5′-OH group in pool 14 RNA.

Mentions: In order to enrich unreported catalytic species from the RNA pool, the selection strategy was built upon three layers of constraints below (Figure 1). The first constraint was to use ATP-γS as a substrate. This reagent had been used in the selection of kinase ribozymes where active RNAs were tagged with a thiophosphate transferred from ATP-γS to its own 5′-OH or internal 2′-OH (4,5). In theory, this reagent should tag any active species that transfer thiophosphate to any sites including the 3′-OH and 5′-ppp (if 5′-ppp-RNA were used) groups, although such ribozymes were not reported previously.Figure 1.


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

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

Selection of 5′-ppp-dependent cis-thiophosphorylating ribozymes from a random pool. 5′-ppp random RNAs were subjected to the reaction with ATP-γS and the resulting thiophosphorylated RNAs were enriched using the following two methods. In round 1–9, active RNAs were isolated on thiopropyl-activated resin. In round 10–14, active RNAs were modified by biotin-iodoacetoamide and isolated on the streptavidin-agarose resin. It should be noted that the combination of these two procedures turned out to be effective for diminishing the appearance of non-specific aptamers that bind to resins. To knock down the population of 5′-ppp-dependent species in pool 14, the counterselection was performed (right); the 5′-ppp group was removed by alkaline phosphatase (AP) to expose the 5′-OH group in pool 14 RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919479&req=5

Figure 1: Selection of 5′-ppp-dependent cis-thiophosphorylating ribozymes from a random pool. 5′-ppp random RNAs were subjected to the reaction with ATP-γS and the resulting thiophosphorylated RNAs were enriched using the following two methods. In round 1–9, active RNAs were isolated on thiopropyl-activated resin. In round 10–14, active RNAs were modified by biotin-iodoacetoamide and isolated on the streptavidin-agarose resin. It should be noted that the combination of these two procedures turned out to be effective for diminishing the appearance of non-specific aptamers that bind to resins. To knock down the population of 5′-ppp-dependent species in pool 14, the counterselection was performed (right); the 5′-ppp group was removed by alkaline phosphatase (AP) to expose the 5′-OH group in pool 14 RNA.
Mentions: In order to enrich unreported catalytic species from the RNA pool, the selection strategy was built upon three layers of constraints below (Figure 1). The first constraint was to use ATP-γS as a substrate. This reagent had been used in the selection of kinase ribozymes where active RNAs were tagged with a thiophosphate transferred from ATP-γS to its own 5′-OH or internal 2′-OH (4,5). In theory, this reagent should tag any active species that transfer thiophosphate to any sites including the 3′-OH and 5′-ppp (if 5′-ppp-RNA were used) groups, although such ribozymes were not reported previously.Figure 1.

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