In general, incorporation of a-L-LNA nucleotides proved to be more efficient replication of a-L-LNA nucleotides, but the results more difficult than incorporation of LNA nucleotides.15-21 presented herein represent the first step toward including a-L- Furthermore, templates containing LNA nucleotides are readily LNA nucleotides in the context of biotechnology, e.g., aptamer used by KOD and 9°Nm for primer extension reactions.17,18 evolution. LNA nucleotides are RNA mimics and have been shown to conformationally steer the 3'-flanking sugar toward N-type sugar Materials and Methods puckers.45 In this light, it can be considered surprising that DNA- mimicking a-L-LNA nucleotides, which do not conformationally 1-(2'-O,4'-C-methylene-a-L-ribofuranosyl)thymine (2) Nucleo- steer flanking nucleotide sugars,24 are poorly accepted by side 17 (0.26 g and 0.45 mmol) was dissolved in methylene polymerases when incorporated into template strands. chloride (5.0 ml) and dichloroacetic acid (0.10 ml and 1.2 mmol) At present, KOD is the polymerase of choice for LNA and Et3SiH (0.15 ml, 0.94 mmol) were slowly added. The nucleotide incorporation.18,19,21 KOD also performed well with reaction was quenched after 1 h using MeOH. The mixture was respect to a-L-LNA nucleotide incorporation though KOD evaporated to dryness. The product was obtained as a white solid could not incorporate consecutive a-L-LNA nucleotides. KOD after column chromatographic purification (0–5% MeOH/DCM, can perform this task when using LNA triphosphates, and even v/v). Yield 0.12 g (100%). 1H NMR data were consistent with extension of primers exclusively using LNA triphosphates has literature.7 This known compound has been synthesized via a new been achieved.21 KOD has been shown to be non-restrictive in route from the O5'-protected precursor. regard to sugar puckering and modification at the 2'-position.18 The O5'-triphosphate of 1-(2'-O,4'-C-methylene-a-L- Accordingly, a-L-LNA TTP was accepted as a substrate by KOD. ribofuranosyl)thymine (a-L-LNA TTP) Nucleoside 2 (0.12 g, ©of nucleotide 9°Nm incorp2orating likewise incorporation. 0consec1utive performed 9°N2mwas a-L-LNA well the with nLucleoatides. only respect polymerase n9°Nm to a-L-LNA dhasalsoewasscooled capable 0.45 sponge mmol) (110 Bto was mg, -i10°oCansdfreschly dissolved 0.51 mmol) in (MeO)3PO was added. idisteilled The (1.9 nPOCl3c(44 reaction ml) and mixture proton eml, . been used for incorporation of LNA nucleotides, and PCR 0.48 mmol) was added dropwise under stirring. The mixture amplification has been achieved using LNA triphosphates and this was stirred for 2 h at temperatures ranging from -10°C to -5°C. polymerase.17 Bu3N (0.32 ml, 1.3 mmol) and a 0.50 M solution of tributyl- Phusion was able to incorporate a-L-LNA nucleotides, but the ammonium pyrophosphate in dimethylformamide (4.0 ml, LNA nucleotides in a Drow had oto be inncorpoorated. tThis wdasalsoisanothter2 rhati-5b°C.The ureacttion ewas q.uenched with a 0.50 M primer could not be extended to full length when several a-L- 2.0 mmol) were added and the reaction mixture was stirred for observed for LNA incorporations, in which case Phusion was able solution of triethylammonium bicarbonate (20 ml). The product to incorporate up to three consecutive LNA-T nucleotides.16 was obtained after gravity column chromatographic purification HIV RT was unsuccessful when we tried to incorporate LNA using a WHATMAN DEAE cellulose-D50 anion-exchange resin nucleotides into oligonucleotides (R. N. Veedu and J. Wengel, and a gradient of triethylammonium bicarbonate in water. 31P unpublished data), however, a-L-LNA nucleotides could be NMR (H2O): d -9.8 (c-P), -10.1 (a-P), -22.4 (β-P). HRMS incorporated under certain conditions. Some full-length product (ESI) m/z calculated for C11H16N2O15P3- (M-): 508.9768; found was observed using template T1 and HIV RT was efficient at 508.9743. incorporating a-L-LNA nucleotides when template T3 was used. General procedure for primer extension experiments. All The incorporation profile of HIV RT was similar to the other water was distilled twice before use. Unmodified primers and three polymerases when templates containing a-L-LNA nucleo- templates were purchased from Sigma-Genosys. Templates tides were used in that full-length extension product was observed containing a-L-LNA nucleotides were produced in-house using for template T6 only and for T4, T5 and T7, extension was commercially available phosphoramidites (Exiqon). Primers were halted at the first site of incorporation or possibly after a 5'-32P labeled by [c-32P]-ATP (~6000 Ci/mmol, GE Healthcare) misincorporation. using T4 polynucleotide kinase (NEB, supplied by Medinova, M0201S) according to a procedure by the manufacturer. Primer Conclusion and template were mixed in a 1:2 ratio. The mixture was heated to 80°C and subsequently slowly cooled to 37°C. Primer exten- KOD, 9°Nm, Phusion and HIV RT polymerases are able to sion reactions were initiated by adding polymerase to a mix of accept a-L-LNA TTP as a substrate and to produce full-length buffer, nucleoside triphosphates and primer:template complex. primer extension reactions. However, primer extension involving The final concentration of nucleoside triphosphates in the used consecutive incorporations of a-L-LNA nucleotides proved mix was approximately 190 mM. After a quick mixing, reaction difficult. Templates containing more than one a-L-LNA nucleo- tubes were incubated at the optimum temperature for the particular tide were not suitable for primer extension reaction when using polymerase. Reaction volumes were 20.0 ml and a 5 ml aliqout was these four polymerases. The fact that standard DNA polymerases added to 2.25 ml loading buffer (95% formamide, 20 mM EDTA, are able to incorporate a-L-LNA nucleotides and to read a-L- bromophenol blue and xylene cyanol dyes) to stop the reaction. LNA-containing templates is notable taking the highly unnatural Products were separated on 13% 7 M urea polyacrylamide gels using conformational and configurational features of a-L-LNA nucleo- TBE buffer (100 mM Tris, 90 mM boric acid, 1 mM EDTA, tides into consideration. Further advances are needed to obtain pH 8.4) and visualized by phosphor imaging. www.landesbioscience.com Artificial DNA: PNA & XNA 19