Development of a Functionally Minimized Mutant of the R3C Ligase Ribozyme Offers Insight into the Plausibility of the RNA World Hypothesis

Kurihara, Eri; Uchida, Sayuri; Umehara, Takuya; Tamura, Koji

doi:10.3390/biology3030452

Cited by 12 publications

(22 citation statements)

References 36 publications

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“…Ligation analysis was performed according to the method of Rogers and Joyce with a slight modification [ 11 , 12 ]. The R3C ligase ribozyme or its variants, dissolved in solution containing 50 mM Tris-HCl (pH 8.5) and 15 mM MgCl 2 , were first heated to 37 °C for 5 min and then cooled to 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…The R3C ribozyme is the smallest among them and was originally composed of 73 nucleotides [ 11 ], in which a three-way junction structure is formed around three stems (P2, P3, P4) ( Figure 1 ). By partially deleting both the “grip” (P4 + P5) and “hammer” (P3) stem-loops, we generated a truncated R3C variant (<∆9–14,GAAA,35–39>, 50 nucleotides) which still retained greater than 10% activity of a full-length R3C ribozyme [ 12 ]. However, a single base-pair deletion from the “grip” region (<∆9–14,GAAA,34–39>, 48 nucleotides) drastically decreased its activity to two orders of magnitude less than that of the ligated product of the full-length R3C ribozyme after 18.5 h at 23 °C ( Figure 2 ) [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

“…By partially deleting both the “grip” (P4 + P5) and “hammer” (P3) stem-loops, we generated a truncated R3C variant (<∆9–14,GAAA,35–39>, 50 nucleotides) which still retained greater than 10% activity of a full-length R3C ribozyme [ 12 ]. However, a single base-pair deletion from the “grip” region (<∆9–14,GAAA,34–39>, 48 nucleotides) drastically decreased its activity to two orders of magnitude less than that of the ligated product of the full-length R3C ribozyme after 18.5 h at 23 °C ( Figure 2 ) [ 12 ]. The acquisition of sufficient activity in ribozymes is considered to be a crucial step in the evolution of the RNA world.…”

Section: Introductionmentioning

confidence: 99%

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The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life

Tanizawa

Uchida

Kurihara

et al. 2018

Biology

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R3C ligase ribozyme catalyzes the nucleophilic attack by a 3′-hydroxyl on a 5′-α-phosphorus of triphosphates to form a 3′-5′-phosphodiester bond. In the present study, although the truncation of R3C ribozyme was accompanied by a large reduction in ligation activity (decrease by two orders of magnitude compared to that of the ligated product of full-length R3C ribozyme after 18.5 h at 23 °C), the introduction of complementary seven-membered kissing-loops served as a “switch” to reactivate the truncated R3C ribozyme with approximately one-fifth of the activity of the full-length R3C ribozyme. This reactivation occurred in a trans-manner, and the grip region and substrate-binding site of the truncated R3C ribozyme were necessary to locate the substrate in the proper position for ligation with the other molecule. Reactivation resulted from complex tertiary interactions between two ribozymes, including kissing-loop interaction-induced annealing and the formation of a stable duplex. The drastic increase of the activity of poorly active ribozymes through the kissing-loop interaction may provide an important clue into the acquisition of substantial activity during the evolution of the RNA world.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life

Tanizawa

Uchida

Kurihara

et al. 2018

Biology

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“…The solution was applied to denaturing 16% polyacrylamide gel for electrophoresis. The gel was analyzed on a Typhoon FLA 7000 (GE Healthcare Japan, Tokyo, Japan) [21].…”

Section: Selective 2'-hydroxyl Acylation Analyzed By Primer Extensionmentioning

confidence: 99%

RNA Aptamers for a tRNA-Binding Protein from Aeropyrum pernix with Homologous Counterparts Distributed Throughout Evolution

Ohmori

Wani

Kitabatake

et al. 2020

Life

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In the present in vitro selection study, we isolated and characterized RNA aptamers for a tRNA-binding protein (Trbp) from an extremophile archaeon Aeropyrum pernix. Trbp-like structures are frequently found not only in aminoacyl-tRNA synthetases but also in diverse types of proteins from different organisms. They likely arose early in evolution and have played important roles in evolution through interactions with key RNA structures. RNA aptamers specific for A. pernix Trbp were successfully selected from a pool of RNAs composed of 60 nucleotides, including a random 30-nucleotide region. From the secondary structures, we obtained a shortened sequence composed of 21 nucleotides, of which the 3′-terminal single stranded CA nucleotides are essential for binding. This may be related to the initial evolutionary role of the universal CCA-3′ terminus of tRNA in the interaction with Trbp-like structures.

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“…This study investigated the ligase ribozyme derived from the R3C ribozyme obtained through in vitro selection [45,46]. This ribozyme catalyzes the formation of a phosphodiester bond between the 5′-triphosphate and 3′-hydroxyl termini of substrate RNAs in the presence of Mg 2+ .…”

Section: Substrate Ligation By Ribozyme and Dnazymementioning

confidence: 99%

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

2017

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Catalytic nucleic acids are regarded as potential therapeutic agents and biosensors. The catalytic activities of nucleic acid enzymes are usually investigated in dilute aqueous solutions, although the physical properties of the reaction environment inside living cells and that in the area proximal to the surface of biosensors in which they operate are quite different from those of pure water. The effect of the molecular environment is also an important focus of research aimed at improving and expanding nucleic acid function by addition of organic solvents to aqueous solutions. In this study, the catalytic activities of RNA and DNA enzymes (hammerhead ribozyme, 17E DNAzyme, R3C ribozyme, and 9DB1 DNAzyme) were investigated using 21 different mixed aqueous solutions comprising organic compounds. Kinetic measurements indicated that these enzymes can display enhanced catalytic activity in mixed solutions with respect to the solution containing no organic additives. Correlation analyses revealed that the turnover rate of the reaction catalyzed by hammerhead ribozyme increased in a medium with a lower dielectric constant than water, and the turnover rate of the reaction catalyzed by 17E DNAzyme increased in conditions that increased the strength of DNA interactions. On the other hand, R3C ribozyme and 9DB1 DNAzyme displayed no significant turnover activity, but their single-turnover rates increased in many mixed solutions. Our data provide insight into the activity of catalytic nucleic acids under various conditions that are applicable to the medical and technology fields, such as in living cells and in biosensors.

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Development of a Functionally Minimized Mutant of the R3C Ligase Ribozyme Offers Insight into the Plausibility of the RNA World Hypothesis

Cited by 12 publications

References 36 publications

The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life

The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life

RNA Aptamers for a tRNA-Binding Protein from Aeropyrum pernix with Homologous Counterparts Distributed Throughout Evolution

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

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