RESEARCH PAPER Scheme 1. Chemical synthesis of a-L-LNA TTP. (i) Cl2CHCOOH, Et3SiH, CH2Cl2 (100%); (ii) (1) (MeO)3PO, proton sponge, POCl3, -10°C A -5°C; (2) tri-n- butylamine, tributylammonium pyrophosphate, dimethylformamide, -5°C; (3) triethylammonium bicarbonate (4%). Results The following seven polymerases were initially explored for their ability to incorporate a-L-LNA nucleotides: the Klenow Synthesis of a-L-LNA TTP. The 5'-triphosphate of the thymine fragment of E. coli DNA polymerase I (A-family polymerase); a-L-LNA nucleoside (a-L-LNA TTP) was synthesized in two KOD, 9°Nm and Phusion DNA polymerases (B-family poly- steps from the known nucleoside 1 (Scheme 1).7 Nucleoside 1 merase); human polymerase β (X-family polymerase); S. solfatari- was detritylated and the resulting nucleoside 27 was converted to cus DNA polymerase IV (Dpo4, Y-family polymerase); and HIV a-L-LNA TTP using the approach initially developed by Ludwig44 RT (reverse transcriptase family polymerase). It was found that and subsequently used by Veedu et al. to synthesize LNA NTPs.16 the four most efficient polymerases for a-L-LNA nucleotide This approach involves phosphorylation of the primary alcohol incorporation were KOD, 9°Nm, Phusion and HIV RT. Figure 3 followed by reaction with pyrophosphate. a-L-LNA TTP was shows the results of primer extension experiments on the T1 ©exchange 2resin (s0eeMat1erials a2ndMethoLdsfor adetailns).destrsated that BKODi,9o°Nm asnd PchusioinDeNA npolymcerases ecan . obtained in an overall yield of 4% after purification over an ion- template for these four polymerases. The experiment demon- Incorporation of a-L-LNA thymine nucleotides. Primer efficiently accept a-L-LNA TTP as a substrate and afford the full- extension experiments were performed on three different length extension products (lane 2). In particular, KOD is very primer-template complexes (Fig. 2) to test the limits of a-L- quick at extending the primer to full length, however accompan- LNA-T nucleotide incorporation across 2'-deoxyadenosines in ied by some product degradation. Although HIV RT could also surrounded by 2'-deoDxynucloeotides of nthe oother tthree nducleo-isdidntot prrogiress btocoumplettion einth.etime the other three the template. Template T1 contained three 2'-deoxyadenosines produce the fully extended product in low yield, the reaction bases, while template T2 contained eight consecutive polymerases required. 2'-deoxyadenosines. Template T3 contained only one 2'- Next, consecutive incorporation of a-L-LNA-T nucleotides deoxyadenosine for coding which was placed at the beginning. was investigated (Fig. 4). KOD, Phusion and HIV RT were Thus, for T3 the polymerases needed to start by extending the unable to extend the primer beyond the first incorporation of a-L- primer with a-L-LNA-T as the first nucleotide. LNA-T (lane 2). 9°Nm DNA polymerase was able to incorporate Positive and negative control reactions were performed in consecutive a-L-LNA-T nucleotides, but full-length extension parallel with reactions with a-L-LNA TTP. The reaction mixture product was not observed. It appears that 9°Nm did not move of the positive controls contained all four natural dNTPs and forward methodically. Rather, 9°Nm seemed to quickly incorpo- led to extension of the primer to full length. Negative control rate several a-L-LNA-T nucleotides before stopping extension. reaction mixtures contained only dATP, dGTP and dCTP and On comparison with a known DNA marker (not shown), we were expected to stop at the first 2'-deoxyadenosine of the could conclude that a major product of the extension was 31 template. a-L-LNA incorporation was tested using reaction nucleotides long corresponding to consecutive incorporation of mixtures containing dATP, dGTP, dCTP and a-L-LNA TTP. five a-L-LNA-T nucleotides, though some of the shorter products Radiolabeled P1 and T1 were used as 19mer and 43mer markers. were also present in trace amounts. Figure 2. Primer-template complexes for primer extension experiments using a-L-LNA TTP. 2’-Deoxyadenosines encoding incorporation of a-L-LNA thymines are underlined. www.landesbioscience.com Artificial DNA: PNA & XNA 15