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A modern mode of activation for nucleic acid enzymes.

Lévesque D, Brière FP, Perreault JP - PLoS ONE (2007)

Bottom Line: One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases.As compared to the allosteric mode of activation, there is no need for the presence of a third partner.In each case, there was a significant gain in terms of substrate specificity.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT
Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

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Related in: MedlinePlus

Catalytic activity of the engineered dependent ligating and capping ribozymes.(A) and (C) are the nucleotide sequences and secondary structures of the ribozyme-substrate complexes of the ligating hairpin and capping ribozymes, respectively. (B) and (D) are autoradiograms of the PAGE gels used to analyse the ligation and capping reactions, respectively. The schematic structures of the nucleic acid enzymes with both the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels. The controls (-) was performed in the absence of ribozyme (lane 1), and, lane 2, in the presence of the unmodified versions. Lane 3 is the ribozyme extended by the blocker sequence. Lanes 4 and 5 are the versions extended by a biosensor that is either complementary, or not, to the substrate, respectively. Finally, lanes 6 and 7 are the on and off ribozymes, respectively. The nucleotide sequences of each engineered nucleic acid enzyme are depicted in Figures S6 and S7.
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pone-0000673-g003: Catalytic activity of the engineered dependent ligating and capping ribozymes.(A) and (C) are the nucleotide sequences and secondary structures of the ribozyme-substrate complexes of the ligating hairpin and capping ribozymes, respectively. (B) and (D) are autoradiograms of the PAGE gels used to analyse the ligation and capping reactions, respectively. The schematic structures of the nucleic acid enzymes with both the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels. The controls (-) was performed in the absence of ribozyme (lane 1), and, lane 2, in the presence of the unmodified versions. Lane 3 is the ribozyme extended by the blocker sequence. Lanes 4 and 5 are the versions extended by a biosensor that is either complementary, or not, to the substrate, respectively. Finally, lanes 6 and 7 are the on and off ribozymes, respectively. The nucleotide sequences of each engineered nucleic acid enzyme are depicted in Figures S6 and S7.

Mentions: Next, we investigated other ribozyme's catalyses. As a first attempt, the ligation catalyzed by the hairpin ribozyme was studied. Extending the 3′-end of the original hairpin ribozyme by 6 nt resulted in the retention of the cleavage product, and consequently favored the reverse reaction (Figure 3A) [14]. Using a 5′-32P-labelled RNA strand possessing a terminal 2′-3′-cyclic phosphate and a second RNA strand possessing a 5′-hydroxyl resulted in the detection of ligation products (Figure 3B). The addition of a biosensor with a sequence complementary to that of the substrate increased the amount of ligation observed. In contrast, the addition of the blocker module almost completely abolished the ligation. Finally, an on version, which includes both the blocker and an appropriate biosensor, exhibited a ligation activity of up to 65%; while an off version (i.e. one with an inappropriate biosensor) showed less than 5% activity.


A modern mode of activation for nucleic acid enzymes.

Lévesque D, Brière FP, Perreault JP - PLoS ONE (2007)

Catalytic activity of the engineered dependent ligating and capping ribozymes.(A) and (C) are the nucleotide sequences and secondary structures of the ribozyme-substrate complexes of the ligating hairpin and capping ribozymes, respectively. (B) and (D) are autoradiograms of the PAGE gels used to analyse the ligation and capping reactions, respectively. The schematic structures of the nucleic acid enzymes with both the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels. The controls (-) was performed in the absence of ribozyme (lane 1), and, lane 2, in the presence of the unmodified versions. Lane 3 is the ribozyme extended by the blocker sequence. Lanes 4 and 5 are the versions extended by a biosensor that is either complementary, or not, to the substrate, respectively. Finally, lanes 6 and 7 are the on and off ribozymes, respectively. The nucleotide sequences of each engineered nucleic acid enzyme are depicted in Figures S6 and S7.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1919428&req=5

pone-0000673-g003: Catalytic activity of the engineered dependent ligating and capping ribozymes.(A) and (C) are the nucleotide sequences and secondary structures of the ribozyme-substrate complexes of the ligating hairpin and capping ribozymes, respectively. (B) and (D) are autoradiograms of the PAGE gels used to analyse the ligation and capping reactions, respectively. The schematic structures of the nucleic acid enzymes with both the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels. The controls (-) was performed in the absence of ribozyme (lane 1), and, lane 2, in the presence of the unmodified versions. Lane 3 is the ribozyme extended by the blocker sequence. Lanes 4 and 5 are the versions extended by a biosensor that is either complementary, or not, to the substrate, respectively. Finally, lanes 6 and 7 are the on and off ribozymes, respectively. The nucleotide sequences of each engineered nucleic acid enzyme are depicted in Figures S6 and S7.
Mentions: Next, we investigated other ribozyme's catalyses. As a first attempt, the ligation catalyzed by the hairpin ribozyme was studied. Extending the 3′-end of the original hairpin ribozyme by 6 nt resulted in the retention of the cleavage product, and consequently favored the reverse reaction (Figure 3A) [14]. Using a 5′-32P-labelled RNA strand possessing a terminal 2′-3′-cyclic phosphate and a second RNA strand possessing a 5′-hydroxyl resulted in the detection of ligation products (Figure 3B). The addition of a biosensor with a sequence complementary to that of the substrate increased the amount of ligation observed. In contrast, the addition of the blocker module almost completely abolished the ligation. Finally, an on version, which includes both the blocker and an appropriate biosensor, exhibited a ligation activity of up to 65%; while an off version (i.e. one with an inappropriate biosensor) showed less than 5% activity.

Bottom Line: One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases.As compared to the allosteric mode of activation, there is no need for the presence of a third partner.In each case, there was a significant gain in terms of substrate specificity.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT
Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

Show MeSH
Related in: MedlinePlus