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• Doessing, H
• Hansen, LH.
• Veedu, RN.
• Wengel, J
• Vester, B
• [+] on Google Scholar
• Doessing, H
• Hansen, LH.
• Veedu, RN.
• Wengel, J
• Vester, B
• [+] on PubMed
• Doessing, H
• Hansen, LH.
• Veedu, RN.
• Wengel, J
• Vester, B

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Molecules 2012, 17(11), 13087-13097; doi:10.3390/molecules171113087

Article

Amplification and Re-Generation of LNA-Modified Libraries

Holger Doessing ^1,2 , Lykke H. Hansen ¹ , Rakesh N. Veedu ^3,4 , Jesper Wengel ³  and Birte Vester ^1,*

¹ Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark ² The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Fremtidsvej 3, 2970 Hoersholm, Denmark ³ Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark ⁴ School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Brisbane, 4072 Queensland, Australia

* Author to whom correspondence should be addressed.

Received: 29 September 2012; in revised form: 31 October 2012 / Accepted: 1 November 2012 / Published: 5 November 2012

(This article belongs to the Special Issue Nucleic Acid Analogs)

Download PDF Full-Text [505 KB, uploaded 5 November 2012 13:08 CET]

Abstract: Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of ‘mock’ in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We observed a decrease in LNA-A content from 20.5% in round 1 to 6.6% in round 7. This decrease was accompanied by a substantial bias against successive LNA-As (poly-LNA adenosine tracts) and a relative over-representation of single LNA-As. Maintaining a library with LNA TTP yielded similar results. Together, these results suggest that dispersed LNA monomers are tolerated in our in vitro selection protocol, and that LNA-modified libraries can be sustained for up to at least seven selection rounds, albeit at reduced levels. This enables the discovery of native LNA aptamers and similar oligonucleotide structures.

Keywords: locked nucleic acid (LNA); in vitro selection; systematic evolution of ligands by exponential enrichment (SELEX); aptamer

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