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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 2. Primer-template complexes for primer extension experiments using α-L-LNA TTP. 2′-Deoxyadenosines encoding incorporation of α-L-LNA thymines are underlined.
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Figure 2: 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.

Mentions: Primer extension experiments were performed on three different primer-template complexes (Fig. 2) 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.


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 2. Primer-template complexes for primer extension experiments using α-L-LNA TTP. 2′-Deoxyadenosines encoding incorporation of α-L-LNA thymines are underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: 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.
Mentions: Primer extension experiments were performed on three different primer-template complexes (Fig. 2) 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.

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