RNA Ligation

Turunen, Janne; Pavlova, L. V.; Hengesbach, Martin; Helm, Mark; Müller, Sabine; Hartmann, Roland K.; Frilander, Mikko J.

doi:10.1002/9783527647064.ch3

Cited by 6 publications

(7 citation statements)

References 72 publications

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“…Pre-orientation of 5′-p and 3′-OH of the RNA substrate is achieved ( a ) by hybridization to a splint. This strategy can be used for ligation with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) and T4 RNA ligase 2 (DNA or RNA splint) ( 68 ); ( b ) by hairpin and linear helper oligonucleotides ( 87 ); ( c ) by a gap splint that upon hybridization leaves the terminal two or three nucleotides of both ends single stranded at the ligation junction ( 88 ); ( d ) by favorable intrinsic structures (i.e. dumbbell folds ( 17 )).…”

Section: Circularization In Vitromentioning

confidence: 99%

“…A number of protocols for enzymatic ligation of synthetic oligonucleotides have been developed ( 68 ). Typically, three different polynucleotide ligases, capable of ligating nicks in single- and/or double-stranded RNA constructs are used: T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1) and T4 RNA ligase 2 (T4 Rnl 2), all encoded in the genome of bacteriophage T4 ( 69 , 70 ).…”

Section: Circularization In Vitromentioning

confidence: 99%

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RNA circularization strategies in vivo and in vitro

Petkovic

Müller

2015

Nucleic Acids Research

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268

218

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In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.

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Section: Circularization In Vitromentioning

confidence: 99%

Section: Circularization In Vitromentioning

confidence: 99%

RNA circularization strategies in vivo and in vitro

Petkovic

Müller

2015

Nucleic Acids Research

Self Cite

268

218

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“…Direct P5 adapter RNA ligation was performed right after the fragmentation step, which is one of the least biased approaches among RNA library preparation methods (13) and also eliminates the need for a second-strand synthesis step before PCR. Since only the terminal positions of the adapter sequence are required to be ribobases (33), we designed DNA adapters with a three-base ribotail (see Table 2) instead of the expensive and less stable RNA adapters normally used for direct RNARNA ligation (5, 13, 34). In order to avoid the potential bias of RNA ligase(s) in terms of base composition of the ligated ends (34, 35), we included two or three degenerate bases in the ribotail as recommended by Jayaprakash et al (34) for miRNA sequencing.…”

Section: Resultsmentioning

confidence: 99%

Digital Gene Expression Analysis with Sample Multiplexing and PCR Duplicate Detection: A Straightforward Protocol

et al. 2016

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Tag-Seq is a high-throughput approach used for discovering SNPs and characterizing gene expression. In comparison to RNA-Seq, Tag-Seq eases data processing and allows detection of rare mRNA species using only one tag per transcript molecule. However, reduced library complexity raises the issue of PCR duplicates, which distort gene expression levels. Here we present a novel Tag-Seq protocol that uses the least biased methods for RNA library preparation combined with a novel approach for joint PCR template and sample labeling. In our protocol, input RNA is fragmented by hydrolysis, and poly(A)-bearing RNAs are selected and directly ligated to mixed DNA-RNA P5 adapters. The P5 adapters contain i5 barcodes composed of sample-specific (moderately) degenerate base regions (mDBRs), which later allow detection of PCR duplicates. The P7 adapter is attached via reverse transcription with individual i7 barcodes added during the amplification step. The resulting libraries can be sequenced on an Illumina sequencer. After sample demultiplexing and PCR duplicate removal with a free software tool we designed, the data are ready for downstream analysis. Our protocol was tested on RNA samples from predator-induced and control Daphnia microcrustaceans.

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“…A small number of strategies, such as click chemistry using strained alkynes, can be applied to both RNA and proteins. and/or in vitro transcribed segments (Solomatin and Herschlag 2009;Turunen et al 2014).…”

Section: Labeling Rna Componentsmentioning

confidence: 99%

“…For short nucleic acids, biotin and digoxigenin can be incorporated during chemical synthesis, whereas the most straightforward strategy for longer RNA is to use biotinylated complementary oligonucleotides that stably hybridize to the RNA of interest, often in added 5 ′ or 3 ′ extensions. For RNA prepared by in vitro transcription using T7 RNA polymerase, modified nucleotides can be incorporated at the 5 ′ end of the transcript ) as well as through enzymatic ligation (Kinoshita et al 1997;Turunen et al 2014) and the conjugation chemistries described above.…”

Section: Modification Of Biomolecules For Surface Immobilizationmentioning

confidence: 99%

Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope

Jalihal

Lund

Walter

2019

Cold Spring Harb Perspect Biol

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RNAs, across their numerous classes, often work in concert with proteins in RNA-protein complexes (RNPs) to execute critical cellular functions. Ensemble-averaging methods have been instrumental in revealing many important aspects of these RNA-protein interactions, yet are insufficiently sensitive to much of the dynamics at the heart of RNP function. Singlemolecule fluorescence microscopy (SMFM) offers complementary, versatile tools to probe RNP conformational and compositional changes in detail. In this review, we first outline the basic principles of SMFM as applied to RNPs, describing key considerations for labeling, imaging, and quantitative analysis. We then sample applications of in vitro and in vivo single-molecule visualization using the case studies of pre-messenger RNA (mRNA) splicing and RNA silencing, respectively. After discussing specific insights single-molecule fluorescence methods have yielded, we briefly review recent developments in the field and highlight areas of anticipated growth.

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RNA Ligation

Cited by 6 publications

References 72 publications

RNA circularization strategies in vivo and in vitro

RNA circularization strategies in vivo and in vitro

Digital Gene Expression Analysis with Sample Multiplexing and PCR Duplicate Detection: A Straightforward Protocol

Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope

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