A Simple and Efficient Method to Transcribe RNAs with Reduced 3′ Heterogeneity

Kao, C. Cheng; Rüdisser, Simon; Zheng, Minxue

doi:10.1006/meth.2000.1131

Cited by 66 publications

(56 citation statements)

References 16 publications

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“…The use of DNA templates ending with C2Ј-methoxy (2Ј-OMe) nucleotide residues, which reduces the 3Ј-end heterogeneity of T7 transcripts (16,25), had no significant effect on the retrohoming efficiency of GG-Lin RNAs synthesized with T3 RNA polymerase and thus appears unnecessary (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation

Zhuang

Mastroianni

White

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3 end of the intron RNA to the 5 end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5 junctions with 5 exon resections, 5 intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.DNA repair ͉ gene targeting ͉ retrotransposition ͉ reverse transcriptase ͉ ribozyme M obile group II introns found in bacterial and organellar genomes are retroelements that consist of an autocatalytic intron RNA and an intron-encoded protein (IEP) with reverse transcriptase (RT) activity (1, 2). The intron RNA and IEP function together in a ribonucleoprotein particle (RNP) to promote the integration of the intron into specific DNA sites by a mechanism in which the intron RNA reverse splices directly into a DNA strand and is then reverse transcribed by the IEP (1). Group II introns use this mechanism both to retrohome to the ligated-exon junction (homing site) in intronless alleles at high frequency and to retrotranspose to ectopic sites that resemble the normal homing site at low frequency. These processes enabled the dispersal of mobile group II introns to diverse bacteria and may have been used for the invasion and proliferation of group II introns in the nuclear genomes of early eukaryotes, where they evolved into spliceosomal introns (1,3,4).Like spliceosomal introns, most group II introns splice via 2 sequential transesterification reactions that result in the formation of an intron lariat RNA (2). For mobile group II introns, the splicing reactions are assisted by the IEP, which binds specifically to the intron RNA to stabilize the catalytically active RNA structure (5). The IEP then remains bound to the excised intron lariat RNA in an RNP that promotes intron mobility (6). RNPs initiate intron mobility by recognizing DNA target sites using both the IEP and base p...

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Section: Resultsmentioning

confidence: 99%

Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation

Zhuang

Mastroianni

White

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…[3] This problem can be reduced by substituting a 2'-methoxyribose sugar for the last two nucleotides (or only the penultimate nucleotide) on the DNA template. [4] Other methods have also been developed for improving the yield and reducing the heterogeneity of RNA produced by in vitro transcription with T7 RNA polymerase. For poorly transcribing RNAs, the addition of a sequence at the 5' end that transcribes more efficiently followed by cleavage of this sequence with template-directed RNase-H can substantially improve the large-scale production of RNA.…”

Section: Synthesis and Purification Of Rna Samples For Nmr Studiesmentioning

confidence: 99%

NMR Methods for Studying the Structure and Dynamics of RNA

et al. 2005

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Proper functioning of RNAs requires the formation of complex three-dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope-labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen-bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long-range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating-frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

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“…The corresponding control reaction with rATP ( Figure 3C, lane 2) showed a minor 25n + 1n product as commonly reported for transcription reactions performed with T7 RNA polymerase. 7 Multiple and successive incorporations of LNA-A nucleotides were likewise investigated using T7 RNA polymerase. For this experiment, we employed a template DNA strand with a segment encoding for continuous incorporation of eight LNA-A nucleotides (T3, Figure 3B).…”

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confidence: 99%

Polymerase Chain Reaction and Transcription Using Locked Nucleic Acid Nucleotide Triphosphates

2008

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Locked nucleic acid (LNA) has demonstrated unique applicability within numerous areas of chemical biology, 1,2 but enzymatic synthesis by DNA and RNA polymerases has been largely unexplored. As a first step in this direction we recently reported preliminary results of primer extension reactions using LNA nucleotide triphosphates. 3,4 As different DNA and RNA polymerases vary in their discrimination of modified nucleotide triphosphates we examined many polymerases and found that Phusion High Fidelity DNA Polymerase was shown to readily accept LNA-TTP and LNA-ATP. We herein apply LNA nucleotide triphosphates to introduce polymerase chain reaction (PCR) amplification of LNAcontaining DNA using 9°N m DNA polymerase and transcription using T7 RNA polymerase.First, we examined the incorporation of LNA-T and LNA-A nucleotides (Figure 1, B ) thymin-1-yl and adenine-9-yl) into a DNA strand from a DNA template using 9°N m DNA polymerase. The template directs six incorporations of LNA-T and LNA-A nucleotides, three of each, mixed with incorporations of DNA-C and DNA-G nucleotides (Figure 2A). A positive control reaction (using all four natural dNTPs) and negative control reaction (with a mixture lacking the nucleotide triphophates similar to the LNA nucleotide triphophates that were to be tested for incorporation) were performed in parallel to the reactions involving the incorporations of LNA monomers. The experiment showed that 9°N m DNA polymerase accepts LNA nucleoside 5′-triphosphates as substrates ( Figure 2C), like we have previously reported for Phusion High Fidelity DNA polymerase. 3 The products were further characterized by MALDI-TOF MS analysis to verify fidelity of LNA incorporations ( Figure S2, Supporting Information). Notably, product degradation was observed after 30 min of reaction in the control reaction using all natural dNTPs ( Figure S3). Such degradation was not observed in the reactions using LNA nucleotides, which we explain as nucleolytic stability induced by the LNA monomers.As the next step we investigated incorporation of LNA-T and LNA-A nucleotides using an LNA-modified DNA template with LNA-nucleotides positioned opposite to the enforced LNA nucleotide incorporation sites. Furthermore, the incorporation of DNA nucleotides opposite to the LNA nucleotides of the template strand was investigated (T4 , Table S1; incorporation of three LNA-T followed by three LNA-A nucleotides mixed with DNA-C and DNA-G nucleotides). This experiment demonstrated that both Phusion High Fidelity DNA polymerase and 9°N m DNA polymerase are capable of incorporating LNA and DNA nucleotides directly opposite to both an LNA or a DNA nucleotide in a template strand and to further extend the strand to full-length ( Figure S4).Encouraged by these results, we investigated the amplification of LNA-modified DNA sequences by PCR using Phusion High Fidelity DNA polymerase and 9°N m DNA polymerase. Template T1 and primers P2 and P3 ( Figure 2B; see Supporting Information for reaction conditions) were designed to enforce incorporat...

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A Simple and Efficient Method to Transcribe RNAs with Reduced 3′ Heterogeneity

Cited by 66 publications

References 16 publications

Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation

Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation

NMR Methods for Studying the Structure and Dynamics of RNA

Polymerase Chain Reaction and Transcription Using Locked Nucleic Acid Nucleotide Triphosphates

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