rtcb  (New England Biolabs)


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    Name:
    RtcB Ligase
    Description:

    Catalog Number:
    M0458
    Price:
    74
    Category:
    Enzymes for Innovation
    Applications:
    Functional Genomics
    Size:
    25 reactions
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    Structured Review

    New England Biolabs rtcb
    RtcB Ligase

    https://www.bioz.com/result/rtcb/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rtcb - by Bioz Stars, 2021-07
    94/100 stars

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    1) Product Images from "ELAC1 Repairs tRNAs Cleaved during Ribosome-Associated Quality Control"

    Article Title: ELAC1 Repairs tRNAs Cleaved during Ribosome-Associated Quality Control

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.01.082

    Requirements of Repairing ANKZF1-Cleaved tRNAs for CCA Addition (A) During ribosome-associated quality control (RQC), stalled ribosomes are dissociated to 60S and 40S ribosomal subunits. The 60S-peptidyl-tRNA complex is recognized by NEMF (teal) and Listerin (orange), which mediates polyubiquitination (Ub) of the nascent protein. ANKZF1 (purple) cleavage of the peptidyl-tRNA generates a ΔCCA tRNA with a 2’,3’-cyclic phosphate (2’,3’ > p) on the discriminator base (N 73 ) that must be resolved before TRNT1 is able to add back the 3’CCA nucleotides (red). (B) A total of 2 ng/μL of in - vitro -transcribed radiolabeled leucyl-tRNA lacking the 3’CCA nucleotides appended to the hepatitis delta virus ribozyme (ΔCCA-HDV) was incubated with nothing to maintain a 2’,3’ > p ( > p), T4 PNK to generate 2’ and 3’ hydroxyl groups, CNP to generate a 2’ phosphate (2’-p), or RtcB to generate a 3’ phosphate (3’-p) on N 73 . The ΔCCA tRNA products were incubated with 100 nM TRNT1 and nucleotides directly (lanes 2–5) or after treatment with calf intestinal alkaline phosphatase (CIP) (lanes 6–8). The reactions were analyzed by SDS-PAGE and autoradiography to assay for the addition of 3’CCA nucleotides producing full-length (FL; red arrowheads) tRNA. Yellow arrowheads denote slower migration of ΔCCA tRNAs without a 2’,3’ > p. (C) Coomassie stain (top) of flow-through (FT) or salt-eluted fractions of reticulocyte lysate from heparin resin. Individual fractions were incubated with radiolabeled ΔCCA-HDV and TRNT1 to assay for FL tRNA production (bottom). Fractions with peak activity (red arrowheads) were pooled for further analyses. (D) Immunoblot of ELAC1 distribution through the fractions shown in (C). .
    Figure Legend Snippet: Requirements of Repairing ANKZF1-Cleaved tRNAs for CCA Addition (A) During ribosome-associated quality control (RQC), stalled ribosomes are dissociated to 60S and 40S ribosomal subunits. The 60S-peptidyl-tRNA complex is recognized by NEMF (teal) and Listerin (orange), which mediates polyubiquitination (Ub) of the nascent protein. ANKZF1 (purple) cleavage of the peptidyl-tRNA generates a ΔCCA tRNA with a 2’,3’-cyclic phosphate (2’,3’ > p) on the discriminator base (N 73 ) that must be resolved before TRNT1 is able to add back the 3’CCA nucleotides (red). (B) A total of 2 ng/μL of in - vitro -transcribed radiolabeled leucyl-tRNA lacking the 3’CCA nucleotides appended to the hepatitis delta virus ribozyme (ΔCCA-HDV) was incubated with nothing to maintain a 2’,3’ > p ( > p), T4 PNK to generate 2’ and 3’ hydroxyl groups, CNP to generate a 2’ phosphate (2’-p), or RtcB to generate a 3’ phosphate (3’-p) on N 73 . The ΔCCA tRNA products were incubated with 100 nM TRNT1 and nucleotides directly (lanes 2–5) or after treatment with calf intestinal alkaline phosphatase (CIP) (lanes 6–8). The reactions were analyzed by SDS-PAGE and autoradiography to assay for the addition of 3’CCA nucleotides producing full-length (FL; red arrowheads) tRNA. Yellow arrowheads denote slower migration of ΔCCA tRNAs without a 2’,3’ > p. (C) Coomassie stain (top) of flow-through (FT) or salt-eluted fractions of reticulocyte lysate from heparin resin. Individual fractions were incubated with radiolabeled ΔCCA-HDV and TRNT1 to assay for FL tRNA production (bottom). Fractions with peak activity (red arrowheads) were pooled for further analyses. (D) Immunoblot of ELAC1 distribution through the fractions shown in (C). .

    Techniques Used: In Vitro, Incubation, SDS Page, Autoradiography, Migration, Staining, Activity Assay

    Related Articles

    Purification:

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: 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. ..

    Ligation:

    Article Title: Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes
    Article Snippet: RNA circularization experiments 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. ..

    Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes
    Article Snippet: RNA circularization experiments 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, 1 to 10 ng of gel-purified retrozyme RNA were ligated using RtcB ligase in its corresponding buffer for 1 h at 37 °C (New England Biolabs). .. Best results for self-ligation assays were obtained with 1 to 10 ng of gel-purified retrozyme RNA in 50 mM Tris–HCl, pH 8, which were firstly denatured at 95 °C for 1 min, slowly cooled down to 25 °C (1 °C decrease every 2 seconds), and then incubated in the presence of 10 to 50 mM MgCl2 for 1h at 25 °C.

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites
    Article Snippet: .. RtcB ligation assays One microlitre 10 μm annealed RNA duplexes (0.5 μm final) were mixed with 2 μL 10× RtcB reaction buffer (New England Biolabs), 1 μL 15 μm RtcB (0.75 μm final; New England Biolabs, M0458S), 2 μL 1 mm GTP, 2 μL 10 mm MnCl2 and 12 μL RNase‐free H2O (total volume = 20 μL) and incubated at 37 °C for 1 h. Samples were analyzed by 15% dPAGE and visualized with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher). .. ResultsIn this study, we aimed to investigate enzymatic ligation as an alternative to cross‐linking in order to capture miRNA‐mRNA interactions, using a new class of miRNA mimics functionalized with 2′, 3′‐cyclic phosphate groups (miRNA > p; Fig. ).

    Incubation:

    Article Title: Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes
    Article Snippet: RNA circularization experiments 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. ..

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites
    Article Snippet: .. RtcB ligation assays One microlitre 10 μm annealed RNA duplexes (0.5 μm final) were mixed with 2 μL 10× RtcB reaction buffer (New England Biolabs), 1 μL 15 μm RtcB (0.75 μm final; New England Biolabs, M0458S), 2 μL 1 mm GTP, 2 μL 10 mm MnCl2 and 12 μL RNase‐free H2O (total volume = 20 μL) and incubated at 37 °C for 1 h. Samples were analyzed by 15% dPAGE and visualized with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher). .. ResultsIn this study, we aimed to investigate enzymatic ligation as an alternative to cross‐linking in order to capture miRNA‐mRNA interactions, using a new class of miRNA mimics functionalized with 2′, 3′‐cyclic phosphate groups (miRNA > p; Fig. ).

    Staining:

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites
    Article Snippet: .. RtcB ligation assays One microlitre 10 μm annealed RNA duplexes (0.5 μm final) were mixed with 2 μL 10× RtcB reaction buffer (New England Biolabs), 1 μL 15 μm RtcB (0.75 μm final; New England Biolabs, M0458S), 2 μL 1 mm GTP, 2 μL 10 mm MnCl2 and 12 μL RNase‐free H2O (total volume = 20 μL) and incubated at 37 °C for 1 h. Samples were analyzed by 15% dPAGE and visualized with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher). .. ResultsIn this study, we aimed to investigate enzymatic ligation as an alternative to cross‐linking in order to capture miRNA‐mRNA interactions, using a new class of miRNA mimics functionalized with 2′, 3′‐cyclic phosphate groups (miRNA > p; Fig. ).

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    New England Biolabs rtcb ligase
    Circularization of Mexican axolotl self-cleaved <t>retrozyme</t> RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with <t>RtcB</t> ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).
    Rtcb Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rtcb ligase/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
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    96
    New England Biolabs rtcb reaction buffer
    <t>RtcB‐mediated</t> ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics. (A) RtcB‐mediated ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) with 8 consecutive and (B) 14 consecutive complementary nt and a variable 5′ overhang of the LMTK3 counter‐strands (0, 2 or 8 nt overhang). Reaction mixtures contained 0.5 μ m <t>RNA</t> duplexes, 0.75 μ m RtcB, 0.1 m m GTP, 1 m m MnCl 2 , and 1x RtcB reaction buffer. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized. Staining with SYBR Gold Nucleic Acid Gel Stain, n = 3, representative gels shown. Full gels shown in Fig. S6 .
    Rtcb Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).

    Journal: bioRxiv

    Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes

    doi: 10.1101/721605

    Figure Lengend Snippet: Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).

    Article Snippet: For the assays of RtcB ligation, 1 to 10 ng of gel-purified retrozyme RNA were ligated using RtcB ligase in its corresponding buffer for 1 h at 37 °C (New England Biolabs).

    Techniques: Purification, Incubation

    Requirements of Repairing ANKZF1-Cleaved tRNAs for CCA Addition (A) During ribosome-associated quality control (RQC), stalled ribosomes are dissociated to 60S and 40S ribosomal subunits. The 60S-peptidyl-tRNA complex is recognized by NEMF (teal) and Listerin (orange), which mediates polyubiquitination (Ub) of the nascent protein. ANKZF1 (purple) cleavage of the peptidyl-tRNA generates a ΔCCA tRNA with a 2’,3’-cyclic phosphate (2’,3’ > p) on the discriminator base (N 73 ) that must be resolved before TRNT1 is able to add back the 3’CCA nucleotides (red). (B) A total of 2 ng/μL of in - vitro -transcribed radiolabeled leucyl-tRNA lacking the 3’CCA nucleotides appended to the hepatitis delta virus ribozyme (ΔCCA-HDV) was incubated with nothing to maintain a 2’,3’ > p ( > p), T4 PNK to generate 2’ and 3’ hydroxyl groups, CNP to generate a 2’ phosphate (2’-p), or RtcB to generate a 3’ phosphate (3’-p) on N 73 . The ΔCCA tRNA products were incubated with 100 nM TRNT1 and nucleotides directly (lanes 2–5) or after treatment with calf intestinal alkaline phosphatase (CIP) (lanes 6–8). The reactions were analyzed by SDS-PAGE and autoradiography to assay for the addition of 3’CCA nucleotides producing full-length (FL; red arrowheads) tRNA. Yellow arrowheads denote slower migration of ΔCCA tRNAs without a 2’,3’ > p. (C) Coomassie stain (top) of flow-through (FT) or salt-eluted fractions of reticulocyte lysate from heparin resin. Individual fractions were incubated with radiolabeled ΔCCA-HDV and TRNT1 to assay for FL tRNA production (bottom). Fractions with peak activity (red arrowheads) were pooled for further analyses. (D) Immunoblot of ELAC1 distribution through the fractions shown in (C). .

    Journal: Cell reports

    Article Title: ELAC1 Repairs tRNAs Cleaved during Ribosome-Associated Quality Control

    doi: 10.1016/j.celrep.2020.01.082

    Figure Lengend Snippet: Requirements of Repairing ANKZF1-Cleaved tRNAs for CCA Addition (A) During ribosome-associated quality control (RQC), stalled ribosomes are dissociated to 60S and 40S ribosomal subunits. The 60S-peptidyl-tRNA complex is recognized by NEMF (teal) and Listerin (orange), which mediates polyubiquitination (Ub) of the nascent protein. ANKZF1 (purple) cleavage of the peptidyl-tRNA generates a ΔCCA tRNA with a 2’,3’-cyclic phosphate (2’,3’ > p) on the discriminator base (N 73 ) that must be resolved before TRNT1 is able to add back the 3’CCA nucleotides (red). (B) A total of 2 ng/μL of in - vitro -transcribed radiolabeled leucyl-tRNA lacking the 3’CCA nucleotides appended to the hepatitis delta virus ribozyme (ΔCCA-HDV) was incubated with nothing to maintain a 2’,3’ > p ( > p), T4 PNK to generate 2’ and 3’ hydroxyl groups, CNP to generate a 2’ phosphate (2’-p), or RtcB to generate a 3’ phosphate (3’-p) on N 73 . The ΔCCA tRNA products were incubated with 100 nM TRNT1 and nucleotides directly (lanes 2–5) or after treatment with calf intestinal alkaline phosphatase (CIP) (lanes 6–8). The reactions were analyzed by SDS-PAGE and autoradiography to assay for the addition of 3’CCA nucleotides producing full-length (FL; red arrowheads) tRNA. Yellow arrowheads denote slower migration of ΔCCA tRNAs without a 2’,3’ > p. (C) Coomassie stain (top) of flow-through (FT) or salt-eluted fractions of reticulocyte lysate from heparin resin. Individual fractions were incubated with radiolabeled ΔCCA-HDV and TRNT1 to assay for FL tRNA production (bottom). Fractions with peak activity (red arrowheads) were pooled for further analyses. (D) Immunoblot of ELAC1 distribution through the fractions shown in (C). .

    Article Snippet: RtcB reactions were performed with 20 ng/μL of radiolabeled ΔCCA-HDV, 0.1 mM GTP, and 0.75 μM RtcB (NEB) in the supplied buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM DTT).

    Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Migration, Staining, Activity Assay

    RtcB‐mediated ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics. (A) RtcB‐mediated ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) with 8 consecutive and (B) 14 consecutive complementary nt and a variable 5′ overhang of the LMTK3 counter‐strands (0, 2 or 8 nt overhang). Reaction mixtures contained 0.5 μ m RNA duplexes, 0.75 μ m RtcB, 0.1 m m GTP, 1 m m MnCl 2 , and 1x RtcB reaction buffer. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized. Staining with SYBR Gold Nucleic Acid Gel Stain, n = 3, representative gels shown. Full gels shown in Fig. S6 .

    Journal: Febs Letters

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites

    doi: 10.1002/1873-3468.13976

    Figure Lengend Snippet: RtcB‐mediated ligation of 2′, 3′‐cyclic phosphate miR‐34a‐5p strands to 5′‐OH mRNA mimics. (A) RtcB‐mediated ligation of miR‐34a‐5p > p (22 nt) to different LMTK3 mRNA mimics (64–72 nt) with 8 consecutive and (B) 14 consecutive complementary nt and a variable 5′ overhang of the LMTK3 counter‐strands (0, 2 or 8 nt overhang). Reaction mixtures contained 0.5 μ m RNA duplexes, 0.75 μ m RtcB, 0.1 m m GTP, 1 m m MnCl 2 , and 1x RtcB reaction buffer. Reference oligonucleotides for the respective ligation products (Controls) were chemically synthesized. Staining with SYBR Gold Nucleic Acid Gel Stain, n = 3, representative gels shown. Full gels shown in Fig. S6 .

    Article Snippet: RtcB ligation assays One microlitre 10 μm annealed RNA duplexes (0.5 μm final) were mixed with 2 μL 10× RtcB reaction buffer (New England Biolabs), 1 μL 15 μm RtcB (0.75 μm final; New England Biolabs, M0458S), 2 μL 1 mm GTP, 2 μL 10 mm MnCl2 and 12 μL RNase‐free H2O (total volume = 20 μL) and incubated at 37 °C for 1 h. Samples were analyzed by 15% dPAGE and visualized with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher).

    Techniques: Ligation, Synthesized, Staining

    Mechanistic details of RtcB‐mediated RNA > p ligation (A) Different mechanisms suggested for bacterial and human RNA > p ligation. Direct ligation between RNA 5′‐OH and 2′, 3′‐cyclic phosphate termini has been suggested in HeLa lysate [ 19 , 37 , 38 ]. For bacterial RtcB, hydrolysis of the 2′, 3′‐cycP to a 3′‐P terminus and subsequent ligation of the 3′‐P terminus has been suggested [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. (B) RtcB‐mediated cyclization of the stem‐loop RNApre‐20 > pin H 2 16 O and (C) H 2 18 O. RtcB ligation was performed at 37 °C for 1 h in the presence of 50 m m Tris/HCl (pH = 8), 1 m m MnCl 2 , 0.1 m m GTP, 0.75 µ m RtcB and 0.5 µ m pre‐20 > p. Ligation was quenched through the addition of Na 2 EDTA to a final concentration of 50 m m . LC‐MS analysis was performed on an Agilent 1200/6130 system fitted with a Waters acquity UPLC OST C‐18 column (2.1 × 50 mm, 1.7 μm) at 65 °C, with a gradient of 5–35% eluent B in 14 min with a flow rate of 0.3 mL·min −1 . Eluent A was aqueous hexafluoroisopropanol (0.4 M) containing triethylamine (15 m m ). Eluent B was methanol. UV trace (260 nm) between 7.5 and 10 min is shown. B = nucleobase.

    Journal: Febs Letters

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites

    doi: 10.1002/1873-3468.13976

    Figure Lengend Snippet: Mechanistic details of RtcB‐mediated RNA > p ligation (A) Different mechanisms suggested for bacterial and human RNA > p ligation. Direct ligation between RNA 5′‐OH and 2′, 3′‐cyclic phosphate termini has been suggested in HeLa lysate [ 19 , 37 , 38 ]. For bacterial RtcB, hydrolysis of the 2′, 3′‐cycP to a 3′‐P terminus and subsequent ligation of the 3′‐P terminus has been suggested [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. (B) RtcB‐mediated cyclization of the stem‐loop RNApre‐20 > pin H 2 16 O and (C) H 2 18 O. RtcB ligation was performed at 37 °C for 1 h in the presence of 50 m m Tris/HCl (pH = 8), 1 m m MnCl 2 , 0.1 m m GTP, 0.75 µ m RtcB and 0.5 µ m pre‐20 > p. Ligation was quenched through the addition of Na 2 EDTA to a final concentration of 50 m m . LC‐MS analysis was performed on an Agilent 1200/6130 system fitted with a Waters acquity UPLC OST C‐18 column (2.1 × 50 mm, 1.7 μm) at 65 °C, with a gradient of 5–35% eluent B in 14 min with a flow rate of 0.3 mL·min −1 . Eluent A was aqueous hexafluoroisopropanol (0.4 M) containing triethylamine (15 m m ). Eluent B was methanol. UV trace (260 nm) between 7.5 and 10 min is shown. B = nucleobase.

    Article Snippet: RtcB ligation assays One microlitre 10 μm annealed RNA duplexes (0.5 μm final) were mixed with 2 μL 10× RtcB reaction buffer (New England Biolabs), 1 μL 15 μm RtcB (0.75 μm final; New England Biolabs, M0458S), 2 μL 1 mm GTP, 2 μL 10 mm MnCl2 and 12 μL RNase‐free H2O (total volume = 20 μL) and incubated at 37 °C for 1 h. Samples were analyzed by 15% dPAGE and visualized with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher).

    Techniques: Ligation, Concentration Assay, Liquid Chromatography with Mass Spectroscopy

    Cross‐linking strategies of miRNAs to their mRNA targets in CLIP protocols. (A) Ligation of miRNA‐mRNA target pairs in miRISC after controlled treatment of the target mRNA with RNase A or T1 during CLASH [ 15 ] or CLEAR‐CLIP [ 14 ] methods; chimeras are formed with low efficiency after 5′‐phosphorylation and T4‐ligase mediated end‐joining. RNA sequencing reveals the miRNA, its mRNA target, and the site of interaction. (B) MiR‐CLIP probes use psoralen to cross‐link to their target mRNAs. MiRNA‐mRNA target pairs are enriched by biotin–streptavidin enrichment [ 6 ]. (C) The proposed protocol (this study). MiRNA‐mRNA chimeras may form with high efficiency after RtcB‐mediated ligation of cyclic phosphate‐terminated miRNA probes to the 5′‐hydroxyl terminus of the target RNA. RNA sequencing reveals the miRNA, its mRNA target, and the site of interaction.

    Journal: Febs Letters

    Article Title: Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites

    doi: 10.1002/1873-3468.13976

    Figure Lengend Snippet: Cross‐linking strategies of miRNAs to their mRNA targets in CLIP protocols. (A) Ligation of miRNA‐mRNA target pairs in miRISC after controlled treatment of the target mRNA with RNase A or T1 during CLASH [ 15 ] or CLEAR‐CLIP [ 14 ] methods; chimeras are formed with low efficiency after 5′‐phosphorylation and T4‐ligase mediated end‐joining. RNA sequencing reveals the miRNA, its mRNA target, and the site of interaction. (B) MiR‐CLIP probes use psoralen to cross‐link to their target mRNAs. MiRNA‐mRNA target pairs are enriched by biotin–streptavidin enrichment [ 6 ]. (C) The proposed protocol (this study). MiRNA‐mRNA chimeras may form with high efficiency after RtcB‐mediated ligation of cyclic phosphate‐terminated miRNA probes to the 5′‐hydroxyl terminus of the target RNA. RNA sequencing reveals the miRNA, its mRNA target, and the site of interaction.

    Article Snippet: RtcB ligation assays One microlitre 10 μm annealed RNA duplexes (0.5 μm final) were mixed with 2 μL 10× RtcB reaction buffer (New England Biolabs), 1 μL 15 μm RtcB (0.75 μm final; New England Biolabs, M0458S), 2 μL 1 mm GTP, 2 μL 10 mm MnCl2 and 12 μL RNase‐free H2O (total volume = 20 μL) and incubated at 37 °C for 1 h. Samples were analyzed by 15% dPAGE and visualized with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher).

    Techniques: Cross-linking Immunoprecipitation, Ligation, RNA Sequencing Assay