An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs

Stark, Martha R.; Pleiss, Jeffrey A.; Deras, Michael L.; Scaringe, Stephen A.; Rader, Stephen D.

doi:10.1261/rna.93506

Cited by 58 publications

(39 citation statements)

References 20 publications

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“…the two genomes). The relatively high occurrence of spike #2 might be due to the identity of its first nucleotide (a cytosine; see Methods) which has been considered a preferred substrate for the T4 RNA ligase that is used for library construction (Romaniuk and Uhlenbeck, 1983;Stark et al, 2006). In conclusion, spike-in #1 appears to be well suited for the given assay system and thus, was added to each of the RNA samples of the pilot experiment (see Fig.…”

Section: Defining the Minimal Materials Requirements For A Dual Rna-sementioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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Section: Defining the Minimal Materials Requirements For A Dual Rna-sementioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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“…Preferentially, the acceptor and the donor are brought together by base-pairing such that the site of ligation is in a hairpin loop [136,139]. However, it has been shown that RNA ligase can also be used in combination with a DNA oligonucleotide annealing with the site of ligation designed to mimic the natural substrate of RNA ligase [140]. To prevent selfligation or ligation of the fragments in the improper sequential order (especially using T4 RNA ligase), the acceptor fragment should contain a hydroxyl group both at its 5' and 3' ends, whereas the donor fragment should have a monophosphate at the ligation site and a monophosphate or a 2',3'-cyclic phosphate at the 3'-end.…”

Section: Selective Isotope Labeling For Larger Rnasmentioning

confidence: 99%

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

Dominguez

Schubert

Duss

et al. 2011

Progress in Nuclear Magnetic Resonance Spectroscopy

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“…This method has been used to generate successfully a wide range of modified RNAs, such as selenium-modified RNAs for X-ray crystallography studies (9); however, the ligation step often takes many hours for completion and/or suffers from poor efficiency, as well as requiring large amounts of DNA ligase. A recent modification of the method employs RNA ligase, modified RNA fragments, and DNA splint to achieve highly efficient and rapid ligation of synthetic RNA fragments (Figure 4, panel d) (62,63). In this case, the RNA fragments are single-stranded (i.e., not paired with the DNA splint) at the ligation site in order to accommodate T4 RNA ligase, which prefers single-stranded substrates.…”

Section: Semisynthesis Approachesmentioning

confidence: 99%

Combined Approaches to Site-Specific Modification of RNA

2008

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Both natural and unnatural modifications in RNA are of interest to biologists and chemists. More than 100 different analogs of the four standard RNA nucleosides have been identified in nature. Unnatural modifications are useful for structure and mechanistic studies of RNA. This Review highlights chemical, enzymatic, and combined (semisynthesis) approaches to generate sitespecifically modified RNAs. The availability of these methods for site-specific modifications of RNAs of all sizes is important in order to study the relationships between RNA chemical composition, structure, and function.RNAs undergo specific post-transcriptional modification by a wide variety of enzymes and ribonucleoprotein complexes (1,2). More than 100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (3). Examples from each of the four major categories of base isomerization, base modification, sugar modification, and hypermodification are shown in Figure 1, panel a. Similarly, a large number of unnatural modifications have been synthesized and used for structure and mechanistic studies of RNA (4-6). A few examples are shown in Figure 1, panel b (7-10). Natural modified nucleotides are often found in the functionally important regions of RNA, such as the peptidyl transferase center of ribosomal RNA (rRNA), the anticodon loop of transfer RNAs, or the branch site of spliceosomal . Several modifications, such as pseudouridine (14), have been known for almost 50 years. Their locations in natural RNAs can, in some cases, be highly conserved; yet, our understanding of their biological roles is still incomplete (15). Through a combination of biological, chemical, and biophysical approaches, much can be learned regarding the roles of modified nucleotides. Similarly, the incorporation of unnatural modifications may be useful in order to study the biological roles and functions of RNA (5).In a number of cases, the enzymes or small nucleolar RNAs (snoRNAs) that are responsible for site-specific RNA modification have been identified (2). Knock-out studies of the individual modifying enzymes or snoRNAs then allow the function of the modification to be deduced. In cases where the enzymes are not known, or the modification is not a natural one, alternative approaches must be considered. Over the past several decades, several chemical methods have been developed to study the effects of modifications in small RNA model systems. These studies have led to newer, more powerful approaches that allow site-specific modification of large RNAs, reconstitution into biological systems, and studies of RNA structure and function. Chemical SynthesisChemical synthesis of RNA allows for site-selective incorporation of modified nucleotides. Five decades ago, Michelson and coworkers synthesized a thymidylate dinucleotide by the *Corresponding author csc@chem.wayne.edu. Figure 2): i) coupling at the 5′ site with a protected phosphoramidite, ii) capping of the unreacted 5′-hydroxyl groups, ...

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An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs

Cited by 58 publications

References 20 publications

Dual RNA-seq of pathogen and host

Dual RNA-seq of pathogen and host

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

Combined Approaches to Site-Specific Modification of RNA

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