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Nucleic Acids Research 2005 33(1):135-142; doi:10.1093/nar/gki149
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Published online 7 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 © Oxford University Press 2005; all rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use permissions, please contact journals.permissions{at}oupjournals.org.

Article

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

Thorarinn Blondal1,2, Audur Thorisdottir1, Unnur Unnsteinsdottir3, Sigridur Hjorleifsdottir1, Arnthor Ævarsson1, Sveinn Ernstsson1, Olafur H. Fridjonsson1, Sigurlaug Skirnisdottir1, Jon Oskar Wheat1, Anna Gudny Hermannsdottir1, Snorri Th. Sigurdsson3, Gudmundur O. Hreggvidsson1,2, Albert Vernon Smith4 and Jakob K. Kristjansson1,*

1 Prokaria Ltd Gylfaflot 5, 112 Reyjavik, Iceland 2 Department of Biology, University of Iceland Sturlugata 7, 101 Reykjavik, Iceland 3 Science Institute, University of Iceland Dunhaga 3, 107, Reykjavik, Iceland 4 GenThor Ltd Gardastraeti 11, 101 Reykjavik, Iceland

*To whom correspondence should be addressed. Tel: +354 5707900; Fax: +354 5707901; Email: jakob.kristjansson{at}prokaria.com

Received August 28, 2004. Revised December 7, 2004. Accepted December 7, 2004.

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

DDBJ/EMBL/GenBack accession no. CQ796353


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