Rtcb ligase

Manufactured by New England Biolabs

Sourced in United States

RtcB ligase is an enzyme that catalyzes the formation of phosphodiester bonds between RNA fragments. It is used in various RNA-related applications, such as the ligation of RNA molecules.

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Lab products found in correlation

Phase lock heavy tubes, by Quantabio (2 mentions) T4 polynucleotide kinase, by New England Biolabs (2 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Rnase inhibitor, by Nacalai Tesque (1 mentions) Zr small rna page recovery kit, by Zymo Research (1 mentions) T4 rna ligase 1, by New England Biolabs (1 mentions) T4 pnk, by New England Biolabs (1 mentions) Rneasy protect bacteria mini kit, by Qiagen (1 mentions)

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Ligases

8 protocols using rtcb ligase

In Lysate Ligation of tiRNAs

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For the ligation of endogenous tiRNAs, tiRNAs were prepared by in lysate ANG digestion [44 (link)] at 100 nM ANG, followed by gel-purification using ZR small-RNA PAGE Recovery Kit (R1070, Zymo Research, Irvine, CA, USA). The purified tiRNAs were first denatured by heating at 90 °C for 2 min, then mixed with 10x Reaction buffer and RNase inhibitor (Nacalai Tesque, 30260-96) followed by incubation at 37 °C for 10 min for annealing. After mixing with GTP, MnCl₂ and 15 pmol of RtcB ligase (M0458, New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instruction, the mixture was incubated at 37 °C for 1 h.
For the ligation of synthetic tiRNAs to examine the generation of chimeric tRNAs, ligation reaction was performed as described above using 40 pmol of 5′-tiRNA and 20 pmol each of two kinds of 3′-tiRNAs. For the ligation of mismatched tiRNAs^Ser, 20 pmol each of 5′-tiRNA^Ser and 3′-tiRNA^Ser were incubated with RtcB at 37 °C for 20 min. The sequences of synthetic tiRNA oligos are shown in Table S3.

Akiyama Y., Takenaka Y., Kasahara T., Abe T., Tomioka Y, & Ivanov P. (2022). RTCB Complex Regulates Stress-Induced tRNA Cleavage. International Journal of Molecular Sciences, 23(21), 13100.

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In vitro Analysis of RNA Retrozyme Ligation

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For in vitro analysis of the tRNA ligation and self-ligation capabilities of RNA retrozymes, monomeric retrozyme RNAs that resulted from double self-cleavage after transcription (either in the presence or in the absence of [α-32P]ATP) of dimeric constructs were purified from 5% polyacrylamide gels under denaturing conditions (8 M urea, 1× TBE). For the assays of RtcB ligation and self-ligation, 1–10 ng of gel-purified radiolabelled retrozyme RNAs, or 1–4 μg for non-radiolabelled RNAs, were firstly denatured at 95°C for 1 min, cooled down to 25°C (either 0.5°C/s or 1°C/min, with similar results), and then incubated either in the presence of RtcB ligase in its corresponding buffer for 1 h at 37°C (New England Biolabs) or in 50 mM Tris–HCl, pH 8 and 10 to 50 mM MgCl₂ for 1 h at 25°C, respectively.

Cervera A, & de la Peña M. (2020). Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes. Nucleic Acids Research, 48(9), 5054-5064.

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Efficient 3' Adapter Ligation of Small RNAs

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15–30 nt sRNAs used for RtcB ligation assay were treated with T4 Pnk (3′ phosphatase minus) to phosphorylate the 5′-OH of sRNAs according to the manufacturer’s instructions. After heat inactivation, the samples were extracted with phenol-chloroform, precipitated with ethanol-NaAc, and dissolved in RNase-free water. RtcB ligation assay was carried out in 20 μl final volume containing 50 ng sRNAs, 20 pmol adapter (OH-RNA8-OH), 2 μl 10 × RtcB reaction buffer, 2 μl 10 mM MnCl₂, 1 μl 10 mM GTP, 15 pmol RtcB ligase (for ligation of 3′-adapter to sRNAs with 3′-P or 3′-cP, New England Biolabs), 20 U recombinant ribonuclease inhibitor and RNase-free water. The mixtures were incubated at 37 °C for 2 h, and the ligation products were analyzed by 15% denatured PAGE.

Lai H., Feng N, & Zhai Q. (2023). Discovery of the major 15–30 nt mammalian small RNAs, their biogenesis and function. Nature Communications, 14, 5796.

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Mouse and Drosophila Poly(A) Tail Assay

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MPAT was performed as previously described^{77 (link)}. In brief, 0.2–2 μg of total mouse or Drosophila RNA was ligated to a linker oligonucleotide at the 3′ end using T4 RNA ligase 1 (New England Biolabs) or RtcB ligase (New England Biolabs) and reverse transcribed using M-MLV reverse transcriptase (Thermo Fisher Scientific) and an oligonucleotide complementary to the linker sequence. Poly(A) tails were PCR amplified using a gene-specific forward primer and the anti-linker oligo, followed by precipitation to remove oligonucleotides and free nucleotides. Labelling was achieved with 5 cycles of a nested PCR on the initial PCR product, using internal end-labelled gene specific oligonucleotides and the anti-linker oligo. PCR products were separated on 6 or 8% high resolution polyacrylamide gels and exposed to PhosphorImager screens. Quick CIP (New England Biolabs) and T4 PNK (New England Biolabs) treatment was performed according to manufacturer’s recommendations, followed by purification of the treated RNA using acid phenol and precipitation with sodium acetate before the ligation to the linker oligonucleotide. Oligonucleotide linker and primers used for the MPAT assay are detailed in Supplementary Data File 2.

Clemente P., Calvo-Garrido J., Pearce S.F., Schober F.A., Shigematsu M., Siira S.J., Laine I., Spåhr H., Steinmetzger C., Petzold K., Kirino Y., Wibom R., Rackham O., Filipovska A., Rorbach J., Freyer C, & Wredenberg A. (2022). ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing. Nature Communications, 13, 5750.

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RNA Purification and Ligation

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After gel purification of autocatalytically cleaved RNA, 300 pmol were treated with T4 polynucleotide kinase (New England Biolabs M0201) according to the manufacturer’s protocol at 37 °C for 30 min and inactivated for 20 min at 65 °C. The products were cleaned by phenol chloroform extraction using heavy phase-lock tubes (Quantabio 2302830). 10 pmol of this purified T4-PNK-treated RNA or of the gel purified RNA was ligated using RtcB Ligase (New England Biolabs M0458) for 1 h at 37 °C.

Litke J.L, & Jaffrey S.R. (2019). Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nature biotechnology, 37(6), 667-675.

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RNA Purification and Ligation

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Litke J.L, & Jaffrey S.R. (2019). Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nature biotechnology, 37(6), 667-675.

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RtcB-mediated Identification of RNA Ends

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Total bacterial RNA was extracted using the Qiagen RNeasy Protect Bacteria mini kit and adapter 5′-NNNNTGGAATTGTCGGGTGCCAAGG-3′ was used in RtcB-mediated ligation reactions to identify 3′-P or 2′,3′-cyclic P RNA ends that would be RtcB targets as described.⁵⁸ (link) An in vitro synthesized RNA with a 3′-P terminus was processed in parallel as a positive control. Commercially available RtcB Ligase (New England Biolabs) was used according to the manufacturer’s instructions and the reactions were subjected to RNA sequencing.

Kotta-Loizou I., Giuliano M.G., Jovanovic M., Schaefer J., Ye F., Zhang N., Irakleidi D.A., Liu X., Zhang X., Buck M, & Engl C. (2022). The RNA repair proteins RtcAB regulate transcription activator RtcR via its CRISPR-associated Rossmann fold domain. iScience, 25(11), 105425.

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RNA Ligation and DNA:RNA Hybrid Cleavage

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Ligation assays were performed using the T4 RNA ligase 1 (T4 Rnl1) or RtcB ligase (New England Biolabs). Reactions were assembled according to the manufacturer's protocols and supplemented with 7.5% PEG 8000 and 40 U RNase inhibitor T4 Rnl1 reactions were incubated at 16°C for 16 h or at 25°C for 2 h. RtcB ligase reactions were incubated for 1 h at 37°C. Apurinic/apyrimidinic Endonuclease 1 (APE1) (New England Biolabs) was used to cleave the phosphodiester backbone of double stranded DNA:RNA hybrids 5′ to abasic sites. RNA ligation products were hybridised with a reverse complementary DNA oligo by heating at 80°C for 5 min and cooling-down to RT. Hybridised samples were supplemented with 1 μl NEBuffer 4 and 5 U APE1 in a total volume of 10 μl. Reactions were incubated at 37°C for 1 h.

Weixler L., Feijs K.L, & Zaja R. (2022). ADP-ribosylation of RNA in mammalian cells is mediated by TRPT1 and multiple PARPs. Nucleic Acids Research, 50(16), 9426-9441.

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