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Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides.

Højland T, Veedu RN, Vester B, Wengel J - Artif DNA PNA XNA (2012 Jan-Mar)

Bottom Line: It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT.Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides.In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

ABSTRACT
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

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Figure 6. Primer-template complexes for primer extension experiments using templates containing α-L-LNA nucleotides. α-L-LNA nucleotides are underlined.
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Figure 6: Figure 6. Primer-template complexes for primer extension experiments using templates containing α-L-LNA nucleotides. α-L-LNA nucleotides are underlined.

Mentions: 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 (Fig. 6). 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.


Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides.

Højland T, Veedu RN, Vester B, Wengel J - Artif DNA PNA XNA (2012 Jan-Mar)

Figure 6. Primer-template complexes for primer extension experiments using templates containing α-L-LNA nucleotides. α-L-LNA nucleotides are underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368812&req=5

Figure 6: Figure 6. Primer-template complexes for primer extension experiments using templates containing α-L-LNA nucleotides. α-L-LNA nucleotides are underlined.
Mentions: 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 (Fig. 6). 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.

Bottom Line: It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT.Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides.In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

ABSTRACT
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

Show MeSH