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RNA synthesis by in vitro selected ribozymes for recreating an RNA world.

Martin LL, Unrau PJ, Müller UF - Life (Basel) (2015)

Bottom Line: The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst.This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms.These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. lyssam@sfu.ca.

ABSTRACT
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme's possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.

No MeSH data available.


Related in: MedlinePlus

Secondary structure comparison of in vitro selected ligase and polymerase ribozymes. (A) The Class I ligase secondary structure; (B) Proposed secondary structure of the B6.61 RNAP ribozyme in complex with its preferred primer-template (blue) and optimal template sequence shown in cyan. Mutations relative to R18 polymerase are boxed in red; (C) Proposed secondary structure of the tC19z RNAP ribozyme in complex with a highly repetitive template sequence (cyan). The 5'-end of the ribozyme was engineered to hybridize to the 3' of the template, promoting longer extension of the primer but also blocking the ribozyme from complete primer extension [17]. Mutations relative to R18 polymerase are boxed in red.
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life-05-00247-f006: Secondary structure comparison of in vitro selected ligase and polymerase ribozymes. (A) The Class I ligase secondary structure; (B) Proposed secondary structure of the B6.61 RNAP ribozyme in complex with its preferred primer-template (blue) and optimal template sequence shown in cyan. Mutations relative to R18 polymerase are boxed in red; (C) Proposed secondary structure of the tC19z RNAP ribozyme in complex with a highly repetitive template sequence (cyan). The 5'-end of the ribozyme was engineered to hybridize to the 3' of the template, promoting longer extension of the primer but also blocking the ribozyme from complete primer extension [17]. Mutations relative to R18 polymerase are boxed in red.

Mentions: The most successful polymerase ribozyme to date was developed in three stages: First, a ligase ribozyme was developed by in vitro selection from a random sequence library containing ~1015 different sequences with 220 randomized nucleotides [15]. This ribozyme, termed the “Class I Ligase” (Figure 6A) catalyzes the nucleophilic attack of 3'-hydroxyl groups on RNA 5'-triphosphates, generating 3'-5'-phosphodiester bonds at a rate about 107-fold above that of the uncatalyzed reaction. Second, variants of this ligase ribozyme were designed to extend an RNA primer by six nucleotides, using nucleoside triphosphates [41]. Importantly, the fidelity of these nucleotide additions was 92%, on average. This is much higher than the fidelity estimated from the stability of Watson-Crick pairing (~40%) [41], implying that the ribozyme recognizes to some extent the geometry of a Watson-Crick base pair between the template strand and the incoming nucleoside triphosphate at the catalytic site [82]. Third, an accessory domain was developed for the polymerase ribozyme by in vitro selection [42]. To do this, a 76-nucleotide long randomized sequence was appended to the 3'-terminus of the ligase domain. After 18 rounds of in vitro selection this library gave rise to the R18 (round 18) polymerase ribozyme, which facilitates the templated primer extension of 14 nucleotides, with an average fidelity of 97%. The R18 ribozyme has been the starting point for reselections which have generated the closely related R18 family: notable members include the B6.61 [16] (Figure 6B) and tC19z RNA polymerase ribozymes [17] (Figure 6C).


RNA synthesis by in vitro selected ribozymes for recreating an RNA world.

Martin LL, Unrau PJ, Müller UF - Life (Basel) (2015)

Secondary structure comparison of in vitro selected ligase and polymerase ribozymes. (A) The Class I ligase secondary structure; (B) Proposed secondary structure of the B6.61 RNAP ribozyme in complex with its preferred primer-template (blue) and optimal template sequence shown in cyan. Mutations relative to R18 polymerase are boxed in red; (C) Proposed secondary structure of the tC19z RNAP ribozyme in complex with a highly repetitive template sequence (cyan). The 5'-end of the ribozyme was engineered to hybridize to the 3' of the template, promoting longer extension of the primer but also blocking the ribozyme from complete primer extension [17]. Mutations relative to R18 polymerase are boxed in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390851&req=5

life-05-00247-f006: Secondary structure comparison of in vitro selected ligase and polymerase ribozymes. (A) The Class I ligase secondary structure; (B) Proposed secondary structure of the B6.61 RNAP ribozyme in complex with its preferred primer-template (blue) and optimal template sequence shown in cyan. Mutations relative to R18 polymerase are boxed in red; (C) Proposed secondary structure of the tC19z RNAP ribozyme in complex with a highly repetitive template sequence (cyan). The 5'-end of the ribozyme was engineered to hybridize to the 3' of the template, promoting longer extension of the primer but also blocking the ribozyme from complete primer extension [17]. Mutations relative to R18 polymerase are boxed in red.
Mentions: The most successful polymerase ribozyme to date was developed in three stages: First, a ligase ribozyme was developed by in vitro selection from a random sequence library containing ~1015 different sequences with 220 randomized nucleotides [15]. This ribozyme, termed the “Class I Ligase” (Figure 6A) catalyzes the nucleophilic attack of 3'-hydroxyl groups on RNA 5'-triphosphates, generating 3'-5'-phosphodiester bonds at a rate about 107-fold above that of the uncatalyzed reaction. Second, variants of this ligase ribozyme were designed to extend an RNA primer by six nucleotides, using nucleoside triphosphates [41]. Importantly, the fidelity of these nucleotide additions was 92%, on average. This is much higher than the fidelity estimated from the stability of Watson-Crick pairing (~40%) [41], implying that the ribozyme recognizes to some extent the geometry of a Watson-Crick base pair between the template strand and the incoming nucleoside triphosphate at the catalytic site [82]. Third, an accessory domain was developed for the polymerase ribozyme by in vitro selection [42]. To do this, a 76-nucleotide long randomized sequence was appended to the 3'-terminus of the ligase domain. After 18 rounds of in vitro selection this library gave rise to the R18 (round 18) polymerase ribozyme, which facilitates the templated primer extension of 14 nucleotides, with an average fidelity of 97%. The R18 ribozyme has been the starting point for reselections which have generated the closely related R18 family: notable members include the B6.61 [16] (Figure 6B) and tC19z RNA polymerase ribozymes [17] (Figure 6C).

Bottom Line: The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst.This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms.These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. lyssam@sfu.ca.

ABSTRACT
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme's possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.

No MeSH data available.


Related in: MedlinePlus