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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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Autoradiograms of cleavage assays performed with controlled cleaving nucleic acid enzymes.(A) (B) and (C) are the reactions performed with the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme, respectively. In each case the structures of the nucleic acid enzyme with the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels, and the control (-) was performed in the absence of any nucleic acid enzyme (lane 1), and, lane 2, in the presence of the unmodified version of the ribozyme. Lane 3 is the version 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. Finally, lanes 6 and 7 are the on and off versions, respectively. The nucleotide sequences of each nucleic acid enzyme are depicted in the Figures S3 to S5.
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pone-0000673-g002: Autoradiograms of cleavage assays performed with controlled cleaving nucleic acid enzymes.(A) (B) and (C) are the reactions performed with the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme, respectively. In each case the structures of the nucleic acid enzyme with the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels, and the control (-) was performed in the absence of any nucleic acid enzyme (lane 1), and, lane 2, in the presence of the unmodified version of the ribozyme. Lane 3 is the version 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. Finally, lanes 6 and 7 are the on and off versions, respectively. The nucleotide sequences of each nucleic acid enzyme are depicted in the Figures S3 to S5.

Mentions: Subsequently, we tried to adapt this mode of activation to other catalytic nucleic acids. As a first attempt, we chose three cleaving molecules: the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme (Figure 2) [9]–[11]. All of these nucleic acid enzymes share a relatively similar architecture. The cleavage site of the substrates is located in the middle of the two stems formed between the catalytic center. The sequences of both of these ribozymes and of the deoxyribozyme have been reported to cleave various substrates that were used [12]–[15]. Each of these catalytic nucleic acids showed significant cleavage of their 5′-end labelled substrates, although at different levels (Figure 2A–C, lane 2). The addition of a blocker domain that “zips off” the binding domain dramatically decreased the cleavage activity (i.e. Figure 2A–C, lane 3). As is observed with the biosensor-blocker HDV ribozyme, the specificity of these engineered nucleic acid molecules was dramatically increased through blocker-mediated inactivation followed by reactivation via an extended complementarity with the appropriate biosensor sequence (Figure 2A–C, lanes 4 to 7). With both the biosensor and blocker domains, the presence of an inappropriate biosensor locked the catalytic core into an off state, while the presence of an appropriate biosensor permitted the switch into the active mode, by displacing the blocker, solely in presence of its specific substrate. The biosensors interact through more base pairs than do the blockers, thereby favoring the on state over the off state. When comparing the kcat/KM' values for the off and on versions of these catalytic molecules, the overall improvements were of a minimum of three orders of magnitude (Table 1). Specifically, the kcat/KM' values vary from 2588-, 2762- and 9167-folds between the off and on version of hammerhead ribozyme, hairpin ribozyme and 10-23 deoxyribozyme, respectively. This illustrates the important gain in terms of specificity. These important differences were mainly the result of dramatic decreases in the kcat values between those of the original structures and those of their respective off states, suggesting that they lost their abilities to perform non-specific cleavage. Importantly, the addition of both a blocker and a biosensor permits the generation of cleaving nucleic acid enzymes that operate in a target-dependent fashion.


A modern mode of activation for nucleic acid enzymes.

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

Autoradiograms of cleavage assays performed with controlled cleaving nucleic acid enzymes.(A) (B) and (C) are the reactions performed with the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme, respectively. In each case the structures of the nucleic acid enzyme with the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels, and the control (-) was performed in the absence of any nucleic acid enzyme (lane 1), and, lane 2, in the presence of the unmodified version of the ribozyme. Lane 3 is the version 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. Finally, lanes 6 and 7 are the on and off versions, respectively. The nucleotide sequences of each nucleic acid enzyme are depicted in the Figures S3 to S5.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919428&req=5

pone-0000673-g002: Autoradiograms of cleavage assays performed with controlled cleaving nucleic acid enzymes.(A) (B) and (C) are the reactions performed with the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme, respectively. In each case the structures of the nucleic acid enzyme with the blocker (red) and biosensor (green) are illustrated above the appropriate lanes of the gels, and the control (-) was performed in the absence of any nucleic acid enzyme (lane 1), and, lane 2, in the presence of the unmodified version of the ribozyme. Lane 3 is the version 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. Finally, lanes 6 and 7 are the on and off versions, respectively. The nucleotide sequences of each nucleic acid enzyme are depicted in the Figures S3 to S5.
Mentions: Subsequently, we tried to adapt this mode of activation to other catalytic nucleic acids. As a first attempt, we chose three cleaving molecules: the hammerhead ribozyme, the 10-23 deoxyribozyme and the hairpin ribozyme (Figure 2) [9]–[11]. All of these nucleic acid enzymes share a relatively similar architecture. The cleavage site of the substrates is located in the middle of the two stems formed between the catalytic center. The sequences of both of these ribozymes and of the deoxyribozyme have been reported to cleave various substrates that were used [12]–[15]. Each of these catalytic nucleic acids showed significant cleavage of their 5′-end labelled substrates, although at different levels (Figure 2A–C, lane 2). The addition of a blocker domain that “zips off” the binding domain dramatically decreased the cleavage activity (i.e. Figure 2A–C, lane 3). As is observed with the biosensor-blocker HDV ribozyme, the specificity of these engineered nucleic acid molecules was dramatically increased through blocker-mediated inactivation followed by reactivation via an extended complementarity with the appropriate biosensor sequence (Figure 2A–C, lanes 4 to 7). With both the biosensor and blocker domains, the presence of an inappropriate biosensor locked the catalytic core into an off state, while the presence of an appropriate biosensor permitted the switch into the active mode, by displacing the blocker, solely in presence of its specific substrate. The biosensors interact through more base pairs than do the blockers, thereby favoring the on state over the off state. When comparing the kcat/KM' values for the off and on versions of these catalytic molecules, the overall improvements were of a minimum of three orders of magnitude (Table 1). Specifically, the kcat/KM' values vary from 2588-, 2762- and 9167-folds between the off and on version of hammerhead ribozyme, hairpin ribozyme and 10-23 deoxyribozyme, respectively. This illustrates the important gain in terms of specificity. These important differences were mainly the result of dramatic decreases in the kcat values between those of the original structures and those of their respective off states, suggesting that they lost their abilities to perform non-specific cleavage. Importantly, the addition of both a blocker and a biosensor permits the generation of cleaving nucleic acid enzymes that operate in a target-dependent fashion.

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