Figure 5. Primer extension using template T3. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of a-L-LNA-T nucleotides (dATP, dGTP, dCTP and a-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); lane 4: P1 (19mer). nucleotide stretch of a-L-LNA nucleotides. In templates T4 and incorporation using template T2 proved to be much more T6, a-L-LNA nucleotides are surrounded by 2'-deoxynucleotides. difficult (Fig. 4). A NMR structure determination of an a-L- Incorporation of 2'-deoxynucleotides using templates contain- LNA/DNA:DNA duplex concluded that the DNA backbone ing a-L-LNA nucleotides was tested by positive control reactions must rearrange to accommodate the a-L-LNA nucleotides in in which the mixture contained all four natural dNTPs. Negative order for optimal Watson-Crick base pairing to take place.24 This ©contained 2only d0GTP, 1dCTP 2anddTTP L(for aincorpnoration dacrosesT1 sand T2. BIntheicaoseof Ts1,the cgrowiing eprimer nstrancddid enot . control reactions were run in parallel. Negative control mixtures may explain the differences in primer extension between templates a-L-LNA-T) or dATP, dCTP and dTTP (for incorporation across contain consecutive a-L-LNA nucleotides, which meant that the a-L-LNA-5-methyl-C). backbone could possibly rearrange to keep the 3'-oxygen in the KOD, 9°Nm, Phusion and HIV RT which performed well at right position in the polymerase active site for further extension. a-L-LNA-T incorporations were investigated for their ability to Primer extension using templates containing a-L-LNA primer extension expeDriments ousing tenmplates oT4-Tt7are dshownisusing ta-Lr-LNiA-cbontainuingtetmpleates. .Only the T6 template, use templates containing a-L-LNA nucleotides. The results of nucleotides. In general, primer extension was difficult when in Figure 7. All four polymerases demonstrated difficulties in which contained a single a-L-LNA-5-methyl-C nucleotide, was extending the primer using templates T4-T7. However, template capable of templating full-length primer extension (Fig. 7). It was T6 which contained a single a-L-LNA-5-methyl-C nucleotide not surprising that templates T5 and T7 could not be used for afforded the full-length extension product by all four polymerases primer extension since the polymerases had great difficulty in (Fig. 7, lane 5) with KOD as the more efficient. incorporating several a-L-LNA nucleotides in a row (Fig. 4). On this note it was, however, surprising that full-length extension Discussion product was not observed for template T4 since a-L-LNA nucleotides were not positioned consecutively. Possibly, Incorporation of a-L-LNA nucleotides. KOD, 9°Nm and rearrangement of the backbone induced by the a-L-LNA Phusion DNA polymerases were able to incorporate a-L-LNA- nucleotides is unfavorable for template function in general, e.g., T nucleotides when template T1 was used (Fig. 3) whereas because of steric clashes between with the polymerase and the Figure 6. Primer-template complexes for primer extension experiments using templates containing a-L-LNA nucleotides. a-L-LNA nucleotides are underlined. www.landesbioscience.com Artificial DNA: PNA & XNA 17