Synthesis of α-L-LNA TTP
The 5′-triphosphate of the thymine α-L-LNA nucleoside (α-L-LNA TTP) was synthesized in two steps from the known nucleoside 1 (Scheme 1
Nucleoside 1 was detritylated and the resulting nucleoside 27
was converted to α-L-LNA TTP using the approach initially developed by Ludwig44
and subsequently used by Veedu et al. to synthesize LNA NTPs.16
This approach involves phosphorylation of the primary alcohol followed by reaction with pyrophosphate. α-L-LNA TTP was obtained in an overall yield of 4% after purification over an ion-exchange resin (see Materials and Methods for details).
Scheme 1. Chemical synthesis of α-L-LNA TTP. (i) Cl2CHCOOH, Et3SiH, CH2Cl2 (100%); (ii) (1) (MeO)3PO, proton sponge, POCl3, -10°C → -5°C; (2) tri-n-butylamine, tributylammonium pyrophosphate, dimethylformamide, -5°C; (more ...)
Incorporation of α-L-LNA thymine nucleotides
Primer extension experiments were performed on three different primer-template complexes () to test the limits of α-L-LNA-T nucleotide incorporation across 2′-deoxyadenosines in the template. Template T1 contained three 2′-deoxyadenosines surrounded by 2′-deoxynucleotides of the other three nucleobases, while template T2 contained eight consecutive 2′-deoxyadenosines. Template T3 contained only one 2′-deoxyadenosine for coding which was placed at the beginning. Thus, for T3 the polymerases needed to start by extending the primer with α-L-LNA-T as the first nucleotide.
Figure 2. Primer-template complexes for primer extension experiments using α-L-LNA TTP. 2′-Deoxyadenosines encoding incorporation of α-L-LNA thymines are underlined.
Positive and negative control reactions were performed in parallel with reactions with α-L-LNA TTP. The reaction mixture of the positive controls contained all four natural dNTPs and led to extension of the primer to full length. Negative control reaction mixtures contained only dATP, dGTP and dCTP and were expected to stop at the first 2′-deoxyadenosine of the template. α-L-LNA incorporation was tested using reaction mixtures containing dATP, dGTP, dCTP and α-L-LNA TTP. Radiolabeled P1 and T1 were used as 19mer and 43mer markers.
The following seven polymerases were initially explored for their ability to incorporate α-L-LNA nucleotides: the Klenow fragment of E. coli DNA polymerase I (A-family polymerase); KOD, 9°Nm and Phusion DNA polymerases (B-family polymerase); human polymerase β (X-family polymerase); S. solfataricus DNA polymerase IV (Dpo4, Y-family polymerase); and HIV RT (reverse transcriptase family polymerase). It was found that the four most efficient polymerases for α-L-LNA nucleotide incorporation were KOD, 9°Nm, Phusion and HIV RT. shows the results of primer extension experiments on the T1 template for these four polymerases. The experiment demonstrated that KOD, 9°Nm and Phusion DNA polymerases can efficiently accept α-L-LNA TTP as a substrate and afford the full-length extension products (lane 2). In particular, KOD is very quick at extending the primer to full length, however accompanied by some product degradation. Although HIV RT could also produce the fully extended product in low yield, the reaction did not progress to completion in the time the other three polymerases required.
Figure 3. Primer extension using template T1. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); (more ...)
Next, consecutive incorporation of α-L-LNA-T nucleotides was investigated (). KOD, Phusion and HIV RT were unable to extend the primer beyond the first incorporation of α-L-LNA-T (lane 2). 9°Nm DNA polymerase was able to incorporate consecutive α-L-LNA-T nucleotides, but full-length extension product was not observed. It appears that 9°Nm did not move forward methodically. Rather, 9°Nm seemed to quickly incorporate several α-L-LNA-T nucleotides before stopping extension. On comparison with a known DNA marker (not shown), we could conclude that a major product of the extension was 31 nucleotides long corresponding to consecutive incorporation of five α-L-LNA-T nucleotides, though some of the shorter products were also present in trace amounts.
Figure 4. Primer extension using template T2. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); (more ...)
We also investigated whether the polymerases needed a running start in order to incorporate α-L-LNA-T nucleotides. We designed template T3 to direct the extension of the primer with α-L-LNA TTP as the first triphosphate to be used as substrate. The results in show that KOD, 9°Nm and HIV RT were able to extend the primer to full length. In fact, KOD was so efficient with template T3 that misincorporation bands are seen in the positive and negative control reactions (, lanes 1 and 3). Phusion DNA polymerase proceeded with difficulty in extending the primer to afford only trace amounts of full-length product.
Figure 5. Primer extension using template T3. Lane 1: positive control (dATP, dGTP, dCTP and TTP); lane 2: incorporation of α-L-LNA-T nucleotides (dATP, dGTP, dCTP and α-L-LNA TTP); lane 3: negative control (dATP, dGTP and dCTP); (more ...)
Primer extension using templates containing α-L-LNA nucleotides
Next, we investigated whether the four polymerases are able to use α-L-LNA TTP as substrate that can tolerate α-L-LNA nucleotides in the template. The commercially available T and 5-methyl-C α-L-LNA phosphoramidites were used to produce templates T4-T7 (). In templates T5 and T7, α-L-LNA nucleotides are placed one after another to produce a four nucleotide stretch of α-L-LNA nucleotides. In templates T4 and T6, α-L-LNA nucleotides are surrounded by 2′-deoxynucleotides.
Figure 6. Primer-template complexes for primer extension experiments using templates containing α-L-LNA nucleotides. α-L-LNA nucleotides are underlined.
Incorporation of 2′-deoxynucleotides using templates containing α-L-LNA nucleotides was tested by positive control reactions in which the mixture contained all four natural dNTPs. Negative control reactions were run in parallel. Negative control mixtures contained only dGTP, dCTP and dTTP (for incorporation across α-L-LNA-T) or dATP, dCTP and dTTP (for incorporation across α-L-LNA-5-methyl-C).
KOD, 9°Nm, Phusion and HIV RT which performed well at α-L-LNA-T incorporations were investigated for their ability to use templates containing α-L-LNA nucleotides. The results of primer extension experiments using templates T4-T7 are shown in . All four polymerases demonstrated difficulties in extending the primer using templates T4-T7. However, template T6 which contained a single α-L-LNA-5-methyl-C nucleotide afforded the full-length extension product by all four polymerases (, lane 5) with KOD as the more efficient.
Figure 7. Primer extension using templates T4-T7. Lanes 1, 3, 5 and 7: positive controls (dATP, dGTP, dCTP and TTP); lanes 2, 4, 6 and 8: negative controls [dGTP, dCTP and dTTP (for incorporation across α-L-LNA-T); or dATP, dCTP and dTTP (more ...)