title: Amplification and re-generation of LNA-modified libraries.
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 &#8216;mock&#8217; 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.
authors.affiliation: Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Holger
authors.initials: H
authors.lastname: Doessing
authors.forename: Lykke H
authors.initials: LH
authors.lastname: Hansen
authors.forename: Rakesh N
authors.initials: RN
authors.lastname: Veedu
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: Birte
authors.initials: B
authors.lastname: Vester
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001482:Base Composition; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D015723:Gene Library; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D013942:Thymine Nucleotides / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D013942:Thymine Nucleotides; C477371:locked nucleic acid; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase