Chez Charles/ tags/ pages tagged ligase

Work in progress.

Ligases are inhibited by high concentrations of ATP (Tessier, Brousserau & Vernet, 1986 and many others).

On T4 RNL1:

On T4 RNL2:

  • Primary paper: Ho et al., 2002.
  • Functional residues analysed by Yin et al., 2003.
  • The truncated Rnl2 was published by Ho et al., 2004.
  • Ligation of RNA to a DNA acceptor is very weak (Yin et al., 2004).
  • The RNA acceptor strand participates to its ligation by promoting adenylylation of the donor, and by promoting the formation of the phosphodiester bond. With DNA, this step is 35 times slower (Nandakumar et al., 2005).
  • The K227Q mutation prevents transfer of the andenylyl residue from the linker to RNA ends, thus prevents unwanted ligation products (Viollet et al., 2011).

DNA and RNA ligases can be used to adenylylate a substrate.

  • McLaughlin, Piel and Graeser (1985) noted that adenylylated RNA is a better substrate for T4 RNA ligase, and that adenylylation efficiency after 6h was C » U ~ A > G.
  • Hoffmann and McLaughlin (1987) used T4 RNL 1 to adenylylate RNA, and noted that ssNpR(A) are good acceptors, and pCpN are good donors.
  • Wang and Unrau (2002) noted that purified recombinant ligase can be partly adenylylated, which blocks the reaction on pre-adenylylated substrates. Pre-incubation with pyrophosphate can displace AMP moiety. Commercial ligases are reported to contain mostly non-adenylylated proteins.
  • Ho, Wang, Lima and Shuman (2004) reported the use of RNL(1-249) on pre-adenylylated RNA in absence of ATP for avoiding concatemerisation.
  • Silverman (2004) reported the use a complementary DNA oligonucleotide (leaving 3-4 protruding 5'ribonucleotides) when adenylylating with T4 RNL 1.
  • Torchia, Takagi and Ho (2008) reported the use of the Methanobacterium RNA ligase (MthRnl) to adenylylate RNA.

Sequence biases

(work in progress)

  • Potapov and coll. (2018) studied in details the tolerance to mismatches of T4 DNA ligase when ligating 3' overhangs. Mismatches were better tolerated with higher GC content and lower temperature. Surprisingly, TNA overhangs were especially hard to ligate.

Methods using ligases:

(works continuously in progress)

  • TACS (terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA) tails DNA with As using the TdT, in order to benefit from the ligase's preference for As (Miura and coll., 2019).
RSS Atom
Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging.

Nucleic Acids Res. 2019 May 22. pii: gkz435. doi:10.1093/nar/gkz435

Miura F, Shibata Y, Miura M, Sangatsuda Y, Hisano O, Araki H, Ito T.

Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging.

Add a A-tail with TdT before ligation, because ligase prefers As.

Posted
A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

Nucleic Acids Res. 2018 Jul 27;46(13):e79. doi:10.1093/nar/gky303

Potapov V, Ong JL, Langhorst BW, Bilotti K, Cahoon D, Canton B, Knight TF, Evans TC Jr, Lohman GJS.

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

TNA overhangs are very inefficient to ligate. However ANT are not harder to ligate than similar mismatches. GC-rich overhangs better tolerate mismatches than AT-rich ones. Increasing temperature reduced mismatched ligations. 5′-G mismatches are much better tolerated than 5′-T mismatches.

Posted
Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.

Torchia C, Takagi Y, Ho CK.

Nucleic Acids Res. 2008 Nov;36(19):6218-27. doi:10.1093/nar/gkn602

Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.

Used in the NEB's 5´ DNA Adenylation Kit. Lacks specificity for RNA and DNA. Optimal at 65 °C, inactivated at 75 °C, and mostly adenylylating at 37 °C.

Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes.

Nucleic Acids Res. 2004 Jun 17;32(11):e86 doi:10.1093/nar/gnh085

Cole K, Truong V, Barone D, McGall G.

Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes.

T4 RNA ligase reaction efficiency improved by PEG and pre-adenylation. Direct labelling method for big quantities: RNA fragmentation, then T4 RNA ligation of biotinylated oligos.

Posted
RNA maps reveal new RNA classes and a possible function for pervasive transcription.

Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, Stadler PF, Hertel J, Hackermüller J, Hofacker IL, Bell I, Cheung E, Drenkow J, Dumais E, Patel S, Helt G, Ganesh M, Ghosh S, Piccolboni A, Sementchenko V, Tammana H, Gingeras TR.

Science. 2007 Jun 8;316(5830):1484-8 doi:10.1126/science.1138341

RNA maps reveal new RNA classes and a possible function for pervasive transcription.

Small RNAs were directly labelled with pCpBiotin. Defines PASRs and PALRs as promoter-associated long or short RNAs.

Unambiguous identification of miRNA:target site interactions by different types of ligation reactions.

Grosswendt S, Filipchyk A, Manzano M, Klironomos F, Schilling M, Herzog M, Gottwein E, Rajewsky N.

Mol Cell. 2014 Jun 19;54(6):1042-1054. doi:10.1016/j.molcel.2014.03.049

Unambiguous identification of miRNA:target site interactions by different types of ligation reactions.

Endogenous activity (speculated to be tRNA ligase) detected in the negative controls.

Posted
Purification of histidine-tagged T4 RNA ligase from E. coli.

Biotechniques. 2002 Dec;33(6):1256-60

Wang QS, Unrau PJ.

Purification of histidine-tagged T4 RNA ligase from E. coli.

De-adenylation of T4 RNA ligase is required prior using adenylated substrates. Incubating with pyrophosphate during some purification steps does the job. Commercial T4 usually contains mostly de-adenylated enzyme. Mesuring reaction efficiency without ATP gives adenylation levels.

Posted
Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

Nucleic Acids Res. 2003 Dec 15;31(24):7247-54 doi:10.1093/nar/gkg914

Blondal T, Hjorleifsdottir SH, Fridjonsson OF, Aevarsson A, Skirnisdottir S, Hermannsdottir AG, Hreggvidsson GO, Smith AV, Kristjansson JK.

Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

Optimal pH between 6 and 7. Enhanced by 25% PEG 6000 and 1 mM HCC.

Posted
Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

Nucleic Acids Res. 2005 Jan 7;33(1):135-42. doi:10.1093/nar/gki149

Blondal T, Thorisdottir A, Unnsteinsdottir U, Hjorleifsdottir S, Aevarsson A, Ernstsson S, Fridjonsson OH, Skirnisdottir S, Wheat JO, Hermannsdottir AG, Sigurdsson ST, Hreggvidsson GO, Smith AV, Kristjansson JK.

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

Preference for circularisation. Optimum : 65-70 °C, 25 µM ATP, pH 7.5-8, 10× more efficient than commercial ones.

Posted
Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins.

Nucleic Acids Res. 2005 Jan 14;33(1):388-99 doi:10.1093/nar/gki174

Englert M, Beier H.

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins.

Ligates 2'-3'P molecules to 5'OH. Has a broad substrate range.

Posted
Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA.

Nat Methods. 2015 Mar;12(3):251-7. doi: 10.1038/nmeth.3259

Koh KD, Balachander S, Hesselberth JR, Storici F.

Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA.

Ligation with Arabidopsis tRNA ligase (AtRNL), that favors self-ligation in circular products, that are enriched by digesting linear molecules. rNMP incorporation frequency may be inversely related to dNTP concentration. More rNMP is incorporated on leading strands.

Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay.

Larman HB, Scott ER, Wogan M, Oliveira G, Torkamani A, Schultz PG

Nucleic Acids Res. 2014 Oct 1;42(14):9146-57. doi: 10.1093/nar/gku636

Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay.

Decoy probes to reduce the number of reads for the most highly expressed genes. Uses T4 Rnl2, which was superior in consistency to splintR. Ligation validated by capillary sequencing. When hybridizing oligonucleotides to a target to form a nicked double strand, annealing is more efficient if the 5′ end at the nick is not phosphorylated.

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase.

J Biol Chem. 2003 Nov 7;278(45):43928-38 doi:10.1074/jbc.M307839200

Sawaya R, Schwer B, Shuman S.

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase.

Trl1 can be separated in three funcionnal polypeptides : a cyclic phosphodiesterase, a A/GTP-dependant polynucleotide kinase, and an ATP-dependant 3′-hydroxyl,2′-phosphate – 5′-phosphate ligase.