Assembly of a functional ribozyme from short oligomers by enhanced non-enzymatic ligation

Mapping Intimacies ◽

10.1101/700229 ◽

2019 ◽

Cited By ~ 2

Author(s):

Lijun Zhou ◽

Derek K. O’Flaherty ◽

Jack W. Szostak

Keyword(s):

Genetic Material ◽

Amino Group ◽

Rna Ligase ◽

Rna Ligation ◽

Enzymatic Ligation ◽

Functional Rna

AbstractThe non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of the first ribozymes, leading to early forms of RNA-based life. However, the reported rate of chemical RNA ligation is extremely slow. Here we show that the rate of ligation can be greatly enhanced by employing a 3′-amino group at the 3′-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications allow the rapid copying of long RNA templates by multi-step ligation of tetranucleotides, as well as the assembly of long oligonucleotides from short template splints. Our work shows that a functional RNA ligase ribozyme can be assembled from relatively short oligonucleotides, demonstrating a transition from non-enzymatic ligation to enzymatic ligation. We suggest that the genomes of primitive protocells could have consisted of relatively easily replicated oligonucleotides as short as 10 to 12 nucleotides in length.

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Uridylate Addition and RNA Ligation Contribute to the Specificity of Kinetoplastid Insertion RNA Editing

Molecular and Cellular Biology ◽

10.1128/mcb.20.22.8447-8457.2000 ◽

2000 ◽

Vol 20 (22) ◽

pp. 8447-8457 ◽

Cited By ~ 62

Author(s):

Robert P. Igo ◽

Setareh S. Palazzo ◽

Moffett L. K. Burgess ◽

Aswini K. Panigrahi ◽

Kenneth Stuart

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Editing Site ◽

Rna Ligase ◽

Correct Number ◽

Guide Rnas ◽

Rna Ligation

ABSTRACT RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5′ fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5′ fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.

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New Deoxyribozymes for the Native Ligation of RNA

Molecules ◽

10.3390/molecules25163650 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3650

Author(s):

Carolin P. M. Scheitl ◽

Sandra Lange ◽

Claudia Höbartner

Keyword(s):

Catalytic Activity ◽

In Vitro Selection ◽

High Ligation ◽

Rna Ligase ◽

Dna Library ◽

Phosphodiester Linkage ◽

Synthetic Accessibility ◽

Rna Ligation ◽

Optimal Incubation

Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5′-adenylated RNA (5′-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3′-5′-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5′-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.

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Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles

Nucleic Acids Research ◽

10.1093/nar/gkz729 ◽

2019 ◽

Vol 47 (17) ◽

pp. 8950-8960 ◽

Cited By ~ 3

Author(s):

Yoko Nomura ◽

Yohei Yokobayashi

Keyword(s):

Genetic Information ◽

Large Scale ◽

Design Synthesis ◽

Laboratory Evolution ◽

Rna Ligase ◽

Highly Active ◽

Selection Experiments ◽

Rna Ligation ◽

Scale Design ◽

Sequence Requirements

Abstract Template-directed RNA ligation catalyzed by an RNA enzyme (ribozyme) is a plausible and important reaction that could have been involved in transferring genetic information during prebiotic evolution. Laboratory evolution experiments have yielded several classes of ligase ribozymes, but their minimal sequence requirements remain largely unexplored. Because selection experiments strongly favor highly active sequences, less active but smaller catalytic motifs may have been overlooked in these experiments. We used large-scale DNA synthesis and high-throughput ribozyme assay enabled by deep sequencing to systematically minimize a previously laboratory-evolved ligase ribozyme. After designing and evaluating >10 000 sequences, we identified catalytic cores as small as 18 contiguous bases that catalyze template-directed regiospecific RNA ligation. The fact that such a short sequence can catalyze this critical reaction suggests that similarly simple or even simpler motifs may populate the RNA sequence space which could have been accessible to the prebiotic ribozymes.

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RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings

RSC Advances ◽

10.1039/c9ra01513b ◽

2019 ◽

Vol 9 (15) ◽

pp. 8620-8627 ◽

Cited By ~ 1

Author(s):

Kai Cheng ◽

Ran An ◽

Yixiao Cui ◽

Yaping Zhang ◽

Xutiange Han ◽

...

Keyword(s):

Efficient Production ◽

Rna Ligase ◽

T4 Rna Ligase ◽

Rna Ligation

T4 Rnl2 ligates ssRNA via nick-like structures, leading to efficient production of versatile RNA rings for various applications.

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Composition of the editing complex of Trypanosoma brucei

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2001.0994 ◽

2002 ◽

Vol 357 (1417) ◽

pp. 71-79 ◽

Cited By ~ 23

Author(s):

K. Stuart ◽

A. K. Panigrahi ◽

A. Schnaufer ◽

M. Drozdz ◽

C. Clayton ◽

...

Keyword(s):

Spectrometric Analysis ◽

Genetic Studies ◽

Rna Ligase ◽

Tandem Mass Spectrometric Analysis ◽

Genes Encoding ◽

Tandem Mass Spectrometric ◽

Rna Ligation ◽

Editing Process ◽

Complex Components

The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy.

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Effect of Unpaired Terminal Nucleotides of Substrate RNAs on the RNA Ligation with T4 RNA Ligase

Bulletin of the Chemical Society of Japan ◽

10.1246/bcsj.64.2978 ◽

1991 ◽

Vol 64 (10) ◽

pp. 2978-2982 ◽

Cited By ~ 1

Author(s):

Naoki Sugimoto ◽

Akiko Matsumura ◽

Keiko Hasegawa ◽

Muneo Sasaki

Keyword(s):

Rna Ligase ◽

T4 Rna Ligase ◽

Rna Ligation

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Functional analysis of the mammalian RNA ligase for IRE1 in the unfolded protein response

Bioscience Reports ◽

10.1042/bsr20160574 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 7

Author(s):

Juthakorn Poothong ◽

Witoon Tirasophon ◽

Randal J. Kaufman

Keyword(s):

Escherichia Coli ◽

Er Stress ◽

Unfolded Protein Response ◽

Mammalian Cells ◽

Unfolded Proteins ◽

Rna Ligase ◽

Unfolded Protein ◽

Rna Ligation ◽

The Unfolded Protein Response ◽

Protein Response

The unfolded protein response (UPR) is a conserved signalling pathway activated on the accumulation of unfolded proteins within the endoplasmic reticulum (ER), termed ER stress. Upon ER stress, HAC1/XBP1 undergoes exon/intron-specific excision by inositol requiring enzyme 1 (IRE1) to remove an intron and liberate the 5′ and 3′ exons. In yeast, the 5′ and 3′ HAC1 exons are subsequently ligated by tRNA ligase (Rlg1p), whereas XBP1 ligation in mammalian cells is catalysed by a recently identified ligase, RtcB. In the present study, RNA ligase activity of the human RtcB (hRtcB) involved in the unconventional splicing of XBP1/HAC1 mRNA was explored in an rlg1-100 mutant yeast strain. Distinct from Escherichia coli RtcB and Rlg1p, expression of hRtcB alone inefficiently complemented HAC1/XBP1 splicing and the hRtcB cofactor (archease) was required to promote enzymatic activity of hRtcB to catalyse RNA ligation.

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The zoonotic potential of bat-borne coronaviruses

Emerging Topics in Life Sciences ◽

10.1042/etls20200097 ◽

2020 ◽

Vol 4 (4) ◽

pp. 365-381

Author(s):

Ny Anjara Fifi Ravelomanantsoa ◽

Sarah Guth ◽

Angelo Andrianiaina ◽

Santino Andry ◽

Anecia Gentles ◽

...

Keyword(s):

Genetic Material ◽

Field Research ◽

Sympatric Species ◽

Animal Origin ◽

Mammalian Host ◽

Past Century ◽

The Past ◽

Novel Host ◽

Horseshoe Bat ◽

Human Infections

Seven zoonoses — human infections of animal origin — have emerged from the Coronaviridae family in the past century, including three viruses responsible for significant human mortality (SARS-CoV, MERS-CoV, and SARS-CoV-2) in the past twenty years alone. These three viruses, in addition to two older CoV zoonoses (HCoV-229E and HCoV-NL63) are believed to be originally derived from wild bat reservoir species. We review the molecular biology of the bat-derived Alpha- and Betacoronavirus genera, highlighting features that contribute to their potential for cross-species emergence, including the use of well-conserved mammalian host cell machinery for cell entry and a unique capacity for adaptation to novel host environments after host switching. The adaptive capacity of coronaviruses largely results from their large genomes, which reduce the risk of deleterious mutational errors and facilitate range-expanding recombination events by offering heightened redundancy in essential genetic material. Large CoV genomes are made possible by the unique proofreading capacity encoded for their RNA-dependent polymerase. We find that bat-borne SARS-related coronaviruses in the subgenus Sarbecovirus, the source clade for SARS-CoV and SARS-CoV-2, present a particularly poignant pandemic threat, due to the extraordinary viral genetic diversity represented among several sympatric species of their horseshoe bat hosts. To date, Sarbecovirus surveillance has been almost entirely restricted to China. More vigorous field research efforts tracking the circulation of Sarbecoviruses specifically and Betacoronaviruses more generally is needed across a broader global range if we are to avoid future repeats of the COVID-19 pandemic.

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