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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 1. Structures of LNA and α-L-LNA nucleotides
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Figure 1: Figure 1. Structures of LNA and α-L-LNA nucleotides

Mentions: The structures of LNA and α-L-LNA (Fig. 1) do not seem to be similar at first glance, but it has been shown that the positioning of atoms important for duplex formation (the O5′ and O3′ atoms of the sugar ring and the N1 atom of the nucleobase) overlay to a large extent in the two nucleotides.4 Given the success of enzymatic incorporation of LNA nucleotides15-21 we therefore speculated that the unusual sugar moiety of α-L-LNA could as well be accepted by some polymerases. Many examples of incorporation of nucleotides with unnatural sugar moieties exist in literature,19,26-33 and a number of studies on stereoisomeric forms of 2′-deoxynucleotide triphosphates have shown that these in many cases act as chain terminators.34-43


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 1. Structures of LNA and α-L-LNA nucleotides
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Figure 1. Structures of LNA and α-L-LNA nucleotides
Mentions: The structures of LNA and α-L-LNA (Fig. 1) do not seem to be similar at first glance, but it has been shown that the positioning of atoms important for duplex formation (the O5′ and O3′ atoms of the sugar ring and the N1 atom of the nucleobase) overlay to a large extent in the two nucleotides.4 Given the success of enzymatic incorporation of LNA nucleotides15-21 we therefore speculated that the unusual sugar moiety of α-L-LNA could as well be accepted by some polymerases. Many examples of incorporation of nucleotides with unnatural sugar moieties exist in literature,19,26-33 and a number of studies on stereoisomeric forms of 2′-deoxynucleotide triphosphates have shown that these in many cases act as chain terminators.34-43

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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