T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

BMC Biotechnology

Research Article

Sebastien Viollet¹  Ryan T Fuchs¹  Fanglei Zhuang¹  Gregory B Robb¹  Daniela B Munafo¹ 

[1] New England Biolabs Inc, 240 County Road, 02143, Ipswich, MA, USA;

关键词: Ligase;    Triple Mutant;    Maltose Binding Protein;    Ligation Reaction;    Ligation Product;   

DOI  :  10.1186/1472-6750-11-72

 received in 2011-05-03, accepted in 2011-07-01,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundT4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA.ResultsWith the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction.ConclusionsOur data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.

【 授权许可】

Unknown   
© Viollet et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.