Generation of a catalytic module on a self-folding RNA

Yoshioka, Wataru; Ikawa, Yoshiya; Jaeger, Luc; Shiraishi, Hideaki; Inoue, Tan

doi:10.1261/rna.7170304

Cited by 26 publications

(24 citation statements)

References 25 publications

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“…The results presented herein illustrate how a natural group I intron is also able to perform 3′,5′ RNA ligation. Together with the earlier findings that ribozymes selected by in vitro evolution from group I introns can catalyze RNA ligation 11,19 , this provides additional evidence that prebiotic replication could have been catalyzed by ribozymes related to group I introns 20 . …”

supporting

confidence: 66%

A natural ribozyme with 3′,5′ RNA ligase activity

Vicens

Cech

2009

Nat Chem Biol

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Using electrophoresis, sequencing and enzymatic digestion, we show that the group I intron from the cyanobacterium Anabaena sp. PCC 7120 catalyzes phosphodiester bond formation using a triphosphate on the 5′-terminal nucleotide, much like protein polymerases and engineered ribozymes. In the process, this ribozyme forms a unique circular RNA that incorporates the exogenous guanosine cofactor added during self-splicing. This finding may have relevance to a prebiotic RNA world and to modern biology.Group I introns self-splice in two chemical steps upon addition of a cofactor that can be guanosine (1) or one of its 5′-phosphorylated forms-guanosine 5′-monophosphate (GMP or pG, 2), guanosine 5′-diphosphate (GDP, 3) or guanosine 5′-triphosphate (GTP, 4) 1 . During the first step, this exogenous guanosine (here called 'αG') binds to a guanosine binding site inherent to the folded RNA structure; it then attacks the phosphorus atom at the 5′ splice site, becoming covalently attached to the 5′ end of the intron through a 3′,5′ linkage (Fig. 1a). A conformational change then brings the conserved guanosine at the 3′ end of the intron (called 'ωG') into the guanosine binding site in place of the αG. During the second chemical step, the free 3′ hydroxyl of the 5′ exon attacks the 3′ splice site, resulting in the formation of a phosphodiester bond that ligates the two exons. The released intron RNA is a linear RNA molecule having a guanosine at both the 5′ end (αG) and the 3′ end (ωG). This intron can undergo further transesterification reactions, thereby forming circles that contain fewer nucleotides than the linear intron 2 (Fig. 1a). A parallel pathway that requires hydrolysis at the 3′ splice site leads to full-length circles (Fig. 1b). The formation of full-length circles is a general property of group I introns that may be linked to intron mobility 3 .We recently assayed for self-splicing activity of 12 group I introns under 6 reaction conditions, in the presence of [α-32 P]GTP (5) as the cofactor 4 . While screening for catalytic activity, an unexpected product was observed for a 249-nucleotide group I intron embedded in a tRNA Leu gene from the cyanobacterium Anabaena sp. PCC 7120 5,6 (Fig. 2a). This 32 Plabeled product had an apparent size around 550-600 nucleotides-larger than the initial unspliced precursor RNA (334 nucleotides) and much larger than the self-spliced linear intron, which was observed (as expected) to be around 250 nucleotides in size. No labeled product of that size could be accounted for by known self-splicing mechanisms.The low electrophoretic mobility of this RNA species suggested that it was either multimeric, circular or branched. In order to test for this, a time-course reaction over two HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscripthours was split and loaded onto two separate polyacrylamide gels, which were run using TBE buffer (1× TBE is 100 mM Tris-base, 83 mM boric acid, 1.0 mM EDTA) at a concentration of either 0.5× or 1× (see Supplementary Methods online) (...

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supporting

confidence: 66%

A natural ribozyme with 3′,5′ RNA ligase activity

Vicens

Cech

2009

Nat Chem Biol

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“…We illustrated this approach using GTP-binding aptamers. The conventional approach -designing pools in the sequence neighborhood of a target molecule (Lau et al 2004;Ohuchi et al 2004;Yoshioka et al 2004), however, does not ensure that the designed pools will cover the structural neighbors of the target molecule, unless sequence mutations are made to localized sequence segments, as is commonly done in many experiments. In contrast, our optimized pool design approach (Appendix), allows enrichment of pools with specific RNA topologies or structures (e.g., tRNA-like 5 3 tree).…”

Section: Discussionmentioning

confidence: 99%

“…To overcome this problem, heuristic approaches have been used to enhance the structural diversity of RNA pools. For example, structured pools have been synthesized by maintaining a constant stem-loop (GTP aptamer selection) (Davis and Szostak 2002) and by introducing random segments in existing RNA structures (e.g., purine nucleotide synthase and domains of group I ribozymes) (Jaeger et al 1999;Ohuchi et al 2002Ohuchi et al , 2004Lau et al 2004;Yoshioka et al 2004). Recent works have also investigated the effects of sequence length (Legiewicz et al 2006) and nucleotide composition (Knight et al 2005) on recovery of specific RNAs.…”

Section: Introductionmentioning

confidence: 99%

A computational proposal for designing structured RNA pools for in vitro selection of RNAs

Kim

Gan

Schlick³

2007

RNA

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Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a ''mixing matrix'' approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

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“…Thus, effective methods should make use of primer sites as part of their strategy for predicting high-complexity structures. One approach to increase the structural complexity is to keep the structure constant and introduce random segments in the proximity of the existing structure (Jaeger et al 1999;Ohuchi et al 2002;Lau et al 2004;Yoshioka et al 2004). Other approaches involve changing sequence length and composition (Knight et al 2005;Legiewicz et al 2006).…”

Section: Problemmentioning

confidence: 99%

Computational approaches toward the design of pools for the in vitro selection of complex aptamers

Luo¹,

McKeague²,

Pitre³

et al. 2010

RNA

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It is well known that using random RNA/DNA sequences for SELEX experiments will generally yield low-complexity structures. Early experimental results suggest that having a structurally diverse library, which, for instance, includes high-order junctions, may prove useful in finding new functional motifs. Here, we develop two computational methods to generate sequences that exhibit higher structural complexity and can be used to increase the overall structural diversity of initial pools for in vitro selection experiments. Random Filtering selectively increases the number of five-way junctions in RNA/DNA pools, and Genetic Filtering designs RNA/DNA pools to a specified structure distribution, whether uniform or otherwise. We show that using our computationally designed DNA pool greatly improves access to highly complex sequence structures for SELEX experiments (without losing our ability to select for common one-way and two-way junction sequences).

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Generation of a catalytic module on a self-folding RNA

Cited by 26 publications

References 25 publications

A natural ribozyme with 3′,5′ RNA ligase activity

A natural ribozyme with 3′,5′ RNA ligase activity

A computational proposal for designing structured RNA pools for in vitro selection of RNAs

Computational approaches toward the design of pools for the in vitro selection of complex aptamers

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