T4 RNA ligase

Reetz et al. described the solid-phase enzymatic synthesis of oligonucleotides on Kieselguhr-PDMA-resins via T4 RNA ligase. Goncomitantly, they found that RNase A selectively cleaves the last bound nucleotide at the ribose sugar leaving a 3, 5 - diphosphorylated ohgomer on the resin, but application in synthesis has not yet been undertaken [22]. 

Despite these clear advantages, the efficiency of splinted RNA ligation reactions catalyzed by T4 DNA ligase is typically limited by the amount of enzyme one can introduce into the reaction. This problem is derived from the poor turnover of T4 DNA ligase on DNA/RNA heteroduplexes. More recently, a variant of T4 RNA ligase, called T4 RNA ligase 2 (Nandakumar et al, 2004), has been identified. This enzyme has a pronounced preference... 

Nandakumar, J., et al. (2004). RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2.J. Biol. Chem. 279, 31337—31347. 

Ho, C. K., and Shuman, S. (2002). Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family ofRNA ligases found in all phylogenetic domains. Proc. Natl. Acad. Sci. USA 99, 12709-12714. 

P-Radiolabeled RNA substrate to be cleaved (prepared by reaction of the RNA with y-32P-ATP and T4 polynucleotide kinase alternatively, 3/-32P-radiolabeling with 32P-pCp and T4 RNA ligase can be used)... 

In the case of X REL1, bacteriophage T4 RNA ligase 2 (T4Rnl2) (57, 58) and human DNA ligase Hip (HsLigllip) (59) are used to check for selectivity of identified inhibitors. 

Transfer-RNA Experiments. Transfer RNAphe was purchased from Sigma and used as received. The RNA was 3 -end labeled with cytidine bisphosphate by use of T4 RNA ligase as described previously (18). The labeled tRNA was purified by polyacrylamide gel electrophoresis. The bands were cut out and the RNA was eluted at room temperature overnight and... 

Heckler TG, Chang LH, Zama Y, Naka T, Chorghade MS, Hecht SM. T4 RNA ligase mediated preparation of novel chemically misacylated tRNAPheS. Biochemistry 1984 23 1468-1473. 

England TE, Uhlenbeck OC. Enzymatic oligoribonucleotide syn- 46. thesis with T4 RNA ligase. Biochemistry 1978 17 2069-2076. 

T4 RNA ligase can introduce fluorescent 3 -O-(5 -phos-phoryldeoxycytidyl) S-bimane phosphorothioate into RNA or DNA (Cosstick et al., 1984). Other methods have been developed for fluorochrome labeling of DNA for automated sequencing, either by labeled primers or dye-terminators (fluorescent ddNTP Applied Biosystems) with DNA polymerase or with terminal dNTP transferase (Trainor and Jensen, 1988). We successfully used fluorescein-dUTP (Boehringer Mannheim, soon available), both for automated sequencing and probe preparation. All common DNA polymerases incorporate this analogue efficiently. 

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