t4 rna ligase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher t4 rna ligase
    Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from  S. cerevisiae  tRNA Phe 5  and the dipeptide-RNA conjugate  6  to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate  8.  ( b ) Without splint  7  only marginal amounts of product  8  were formed; reaction conditions: T4 RNA ligase (0.5 U/µl;  c RNA  = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint  7  resulted in 75% yield of  8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl;  c RNA  = 40 µM each strand;  c DNA  = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see  Supplementart Data .
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications"

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq508

    Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from  S. cerevisiae  tRNA Phe 5  and the dipeptide-RNA conjugate  6  to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate  8.  ( b ) Without splint  7  only marginal amounts of product  8  were formed; reaction conditions: T4 RNA ligase (0.5 U/µl;  c RNA  = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint  7  resulted in 75% yield of  8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl;  c RNA  = 40 µM each strand;  c DNA  = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see  Supplementart Data .
    Figure Legend Snippet: Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from S. cerevisiae tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint 7 resulted in 75% yield of 8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c RNA = 40 µM each strand; c DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography

    Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from  E. coli  tRNA Phe 5  and the dipeptide-RNA conjugate  6  to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate  8.  ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl;  c RNA  = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of  8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see  Supplementary Data .
    Figure Legend Snippet: Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from E. coli tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of 8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography, Purification, Liquid Chromatography, Mass Spectrometry

    2) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal:

    doi: 10.1261/rna.5247704

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PPi (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PPi (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    3) Product Images from "Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA"

    Article Title: Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09403-x

    NrnA is a bi-directional exoribonuclease. ( A ) NrnA activity on 3-mer RNA oligonucleotides. WT NrnA activity is compared on *pA3, *pA3-2′ O- me with only the 3′ nucleotide methylated, and *pA3p, radiolabeled at their 5′ ends with  32 P (*) using T4 polynucleotide kinase and [γ 32 P]-ATP. Equivalent amounts of NrnA (0.285 μM) and substrate (1 μM) were used in all reactions. ( B ) NrnA activity on 12-mer RNA oligonucleotides. ( left ) WT NrnA (2.85 μM) was assayed on A12 (1 μM) labeled at its 5′ end. WT ( center ) and R262A R264A ( right ) NrnA (2.85 μM) were assayed on A12 (1 μM) labeled at its 3′ end with *pCp by T4 RNA ligase. ( C ) WT (0.1 μM) and R262A R264A (0.1 μM) NrnA were compared on *A4 (5 μM). Black dividing lines demarcate distinct gels or sections of a gel.
    Figure Legend Snippet: NrnA is a bi-directional exoribonuclease. ( A ) NrnA activity on 3-mer RNA oligonucleotides. WT NrnA activity is compared on *pA3, *pA3-2′ O- me with only the 3′ nucleotide methylated, and *pA3p, radiolabeled at their 5′ ends with 32 P (*) using T4 polynucleotide kinase and [γ 32 P]-ATP. Equivalent amounts of NrnA (0.285 μM) and substrate (1 μM) were used in all reactions. ( B ) NrnA activity on 12-mer RNA oligonucleotides. ( left ) WT NrnA (2.85 μM) was assayed on A12 (1 μM) labeled at its 5′ end. WT ( center ) and R262A R264A ( right ) NrnA (2.85 μM) were assayed on A12 (1 μM) labeled at its 3′ end with *pCp by T4 RNA ligase. ( C ) WT (0.1 μM) and R262A R264A (0.1 μM) NrnA were compared on *A4 (5 μM). Black dividing lines demarcate distinct gels or sections of a gel.

    Techniques Used: Activity Assay, Methylation, Labeling

    4) Product Images from "Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications"

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq508

    Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from  S. cerevisiae  tRNA Phe 5  and the dipeptide-RNA conjugate  6  to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate  8.  ( b ) Without splint  7  only marginal amounts of product  8  were formed; reaction conditions: T4 RNA ligase (0.5 U/µl;  c RNA  = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint  7  resulted in 75% yield of  8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl;  c RNA  = 40 µM each strand;  c DNA  = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see  Supplementart Data .
    Figure Legend Snippet: Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from S. cerevisiae tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint 7 resulted in 75% yield of 8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c RNA = 40 µM each strand; c DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography

    Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from  E. coli  tRNA Phe 5  and the dipeptide-RNA conjugate  6  to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate  8.  ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl;  c RNA  = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of  8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see  Supplementary Data .
    Figure Legend Snippet: Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from E. coli tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of 8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography, Purification, Liquid Chromatography, Mass Spectrometry

    5) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal:

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    6) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal:

    doi: 10.1261/rna.5247704

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PPi (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PPi (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    7) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal:

    doi: 10.1261/rna.5247704

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PPi (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PPi (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    8) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal:

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule
    Figure Legend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Techniques Used: Radioactivity, Ligation

    9) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal:

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    10) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal:

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    11) Product Images from "The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics"

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics

    Journal:

    doi: 10.1002/chem.201302188

    Optimized enzymatic ligation of phosphonated, methylated, and/or phosphorylated aminoacylamino-tRNASec by using three synthetic fragments and T4 RNA ligase, exemplified for Abu(p)-tRNASec. A) Concept for enzymatic ligation of tRNAs with a large variable
    Figure Legend Snippet: Optimized enzymatic ligation of phosphonated, methylated, and/or phosphorylated aminoacylamino-tRNASec by using three synthetic fragments and T4 RNA ligase, exemplified for Abu(p)-tRNASec. A) Concept for enzymatic ligation of tRNAs with a large variable

    Techniques Used: Ligation, Methylation

    Related Articles

    Sequencing:

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: Equimolar amounts of three chemically synthesized tRNA fragments (see for sequence, for 5′-phosphate modification, and ) were dissolved in water (2 /3 of the final total reaction volume, final RNA concentration of 40 μM for each strand). .. The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

    Ligation:

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications
    Article Snippet: Paragraph title: Ligation without DNA splint ... The sample was vortexed and centrifuged before it was treated with T4 RNA ligase (Fermentas; 10 U/µl in storage solution) to give a concentration of 0.5 U/µl (in the final reaction volume).

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: Paragraph title: Enzymatic ligation of tRNASec ... The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

    Synthesized:

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: Equimolar amounts of three chemically synthesized tRNA fragments (see for sequence, for 5′-phosphate modification, and ) were dissolved in water (2 /3 of the final total reaction volume, final RNA concentration of 40 μM for each strand). .. The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

    Purification:

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer). .. The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

    Concentration Assay:

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications
    Article Snippet: One-tenth of the final reaction volume of reaction buffer [500 mM HEPES–NaOH (pH 8.0), 100 mM MgCl2 , 100 mM DTT = ‘10× ligation buffer’], ATP and BSA from stock solutions were added to the mixture to give concentrations of 1 mM ATP and 0.1 mg/ml BSA (in the final reaction volume). .. The sample was vortexed and centrifuged before it was treated with T4 RNA ligase (Fermentas; 10 U/µl in storage solution) to give a concentration of 0.5 U/µl (in the final reaction volume). .. Equimolar amounts of acceptor, donor and DNA strands (final concentration = 40 µM each strand) were mixed in nanofiltered water (about two-third of the final total reaction volume), the solution was heated to 90°C for 2 min and then allowed to cool to room temperature within 15 min.

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications
    Article Snippet: One-tenth of the final reaction volume of reaction buffer [500 mM HEPES–NaOH (pH 8.0), 100 mM MgCl2 , 100 mM DTT = ‘10× ligation buffer’], ATP and BSA from stock solutions were added to the mixture to give concentrations of 0.5 mM ATP and 0.1 mg/ml BSA (in the final reaction volume). .. The sample was vortexed and centrifuged before it was treated with T4 RNA ligase (Fermentas; 10 U/µl in storage solution) to give a concentration of 0.25 U/µl (in the final reaction volume). .. The reaction mixture was extracted twice with an equal volume of a phenol/chloroform/isoamyl alcohol solution (25/24/1, v/v/v) and twice with an equal volume of chloroform/isoamyl alcohol solution (24/1, v/v).

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: Equimolar amounts of three chemically synthesized tRNA fragments (see for sequence, for 5′-phosphate modification, and ) were dissolved in water (2 /3 of the final total reaction volume, final RNA concentration of 40 μM for each strand). .. The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

    Incubation:

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer). .. The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

    Modification:

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics
    Article Snippet: Equimolar amounts of three chemically synthesized tRNA fragments (see for sequence, for 5′-phosphate modification, and ) were dissolved in water (2 /3 of the final total reaction volume, final RNA concentration of 40 μM for each strand). .. The reaction mixture was vortexed and centrifuged before it was treated with T4 RNA ligase (0.4 UμL−1 , Fermentas, 10 UμL−1 in storage buffer).

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  • 92
    Thermo Fisher t4 rna ligase buffer
    Preparation and analysis on circular RNA  in vitro . (A) Schematic of  in vitro  circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the  in vitro  ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient  in vitro  ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C)  In vitro  ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).
    T4 Rna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/Thermo Fisher
    Average 92 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-01
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    94
    Thermo Fisher dna ligation solution
    Preparation and analysis on circular RNA  in vitro . (A) Schematic of  in vitro  circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the  in vitro  ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient  in vitro  ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C)  In vitro  ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).
    Dna Ligation Solution, supplied by Thermo Fisher, 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/dna ligation solution/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna ligation solution - by Bioz Stars, 2020-01
    94/100 stars
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    Preparation and analysis on circular RNA  in vitro . (A) Schematic of  in vitro  circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the  in vitro  ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient  in vitro  ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C)  In vitro  ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Journal: RNA Biology

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    doi: 10.1080/15476286.2018.1435248

    Figure Lengend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Article Snippet: Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C.

    Techniques: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Journal:

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    doi: 10.1039/c7an00670e

    Figure Lengend Snippet: Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Article Snippet: Specifically, 1 nmole of 5′-phosphorylated 3′ adaptor was mixed with 40 μl 50% PEG 8000 (BioLabs), 10 μl 1x T4 RNA ligase reaction buffer, and 5 μl T4 RNA ligase 1 (Thermo Fisher EL0021) in a total volume of 100 μl and reacted at room temperature overnight.

    Techniques: Lysis, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Selection, Gel Purification

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Journal:

    Article Title: Synthesis and Labeling of RNA In Vitro

    doi: 10.1002/0471142727.mb0415s102

    Figure Lengend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Article Snippet: 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase.

    Techniques: Radioactivity, Ligation