Artificial DNA: PNA & XNA 3:1, 14–21; January/February/March 2012; G 2012 Landes Bioscience Enzymatic synthesis of DNA strands containing a-L-LNA (a-L-configured locked nucleic acid) thymine nucleotides Torben Højland,1 Rakesh N. Veedu,1,2 Birte Vester1 and Jesper Wengel1,* 1Department of Physics, Chemistry and Pharmacy; Nucleic Acid Center; Department of Biochemistry and Molecular Biology; University of Southern Denmark; Odense, Denmark; 2School of Chemistry and Molecular Biosciences; The University of Queensland; Brisbane, Australia Keywords: a-L-LNA, polymerase, enzymatic synthesis, modified nucleotide, triphosphate Abbreviations: a-L-LNA, a-L-configured locked nucleic acid; HIV RT, human immunodeficiency virus reverse transcriptase; LNA, locked nucleic acid; PCR, polymerase chain reaction; DCM, dichloromethane; DEAE, diethylaminoethyl; HRMS, high resolution mass spectrometry; ESI, electrospray ionization; TBE, tris-borate-EDTA; EDTA, ethylenediaminetetraacetic acid; Dpo4, DNA polymerase IV; Tris, tris(hydroxymethyl)aminomethane © 52’-tripho0sphate 1ofa-L-2LNA is a sLubstraatefor nthe DNdApolyemerassesKOD, B9°Nm, Pihusoionand sHIV cRT.Thiree edispernsed a-cL-e. We describe the first enzymatic incorporation of an a-L-LNA nucleotide into an oligonucleotide. It was found that the LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of a-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one a-L-LNA nucleotide. DIntrooductionnot disand tC2'-rmodiificbation usince tribeonucle.otides could also be incorporated by KOD.18 Locked nucleic acid1-3 (LNA) and its diastereomer a-L-LNA4-10 The structures of LNA and a-L-LNA (Fig. 1) do not seem to (a-L-configured locked nucleic acid) have found numerous be similar at first glance, but it has been shown that the applications within the field of nucleic acid chemical biology.11-14 positioning of atoms important for duplex formation (the O5' and Recently, enzymatic incorporation of LNA nucleotides has been O3' atoms of the sugar ring and the N1 atom of the nucleobase) realized.15-21 So far, no report on enzymatic incorporation of an overlay to a large extent in the two nucleotides.4 Given the success a-L-LNA nucleotide, or any other LNA stereoisomeric nucleo- of enzymatic incorporation of LNA nucleotides15-21 we therefore tide, has been published. In this paper we disclose the results speculated that the unusual sugar moiety of a-L-LNA could as of initial experiments on the compatibility of polymerases with well be accepted by some polymerases. Many examples of a-L-LNA nucleotides. incorporation of nucleotides with unnatural sugar moieties exist in As is the case with LNA, oligonucleotides containing a-L-LNA literature,19,26-33 and a number of studies on stereoisomeric forms nucleotides show very efficient binding to complementary nucleic of 2'-deoxynucleotide triphosphates have shown that these in acids.4-10 In addition, a-L-LNA nucleotides provide protection many cases act as chain terminators.34-43 from nucleases when incorporated into oligonucleotides.9,22,23 NMR studies concluded that DNA strands containing three a-L- LNA incorporations formed duplexes with DNA and RNA that were of the B-type and intermediate A/B-type, respectively.24,25 Thus, a-L-LNA can be considered a DNA mimic. The interesting properties of a-L-LNA led us to investigate the compatibility of polymerases with a-L-LNA nucleotides. We were encouraged by the fact that LNA nucleotides can be incorporated by a variety of polymerases.15-21 In particular, KOD is very efficient at incorporating LNA nucleotides as well as reading LNA-containing templates.18 In general, KOD was found to be non-restrictive with respect to both furanose ring puckering Figure 1. Structures of LNA and a-L-LNA nucleotides *Correspondence to: Jesper Wengel; Email: jwe@sdu.dk Submitted: 11/21/11; Revised: 01/06/12; Accepted: 01/06/12 http://dx.doi.org/10.4161/adna.19272 14 Artificial DNA: PNA & XNA Volume 3 Issue 1