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Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA.

Lin N, Yan J, Huang Z, Altier C, Li M, Carrasco N, Suyemoto M, Johnston L, Wang S, Wang Q, Fang H, Caton-Williams J, Wang B - Nucleic Acids Res. (2007)

Bottom Line: We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization.We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA.DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Center for Biotechnology and Drug Design, Campus Box 4098, Georgia State University, Atlanta, GA 30302-4098, USA.

ABSTRACT
The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.

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Primer extension using the full-length DNA and boronic acid-labeled DNA as templates. Each 50 µl reaction was performed with 1.2 µM primers 1 and 2/template, 0.25 mM of each dNTPs, 0.25 mM of labeled-TTP (B-TTP), and 3.5 units of high-fidelity DNA polymerase (Roche, Indianapolis, Ind.) under conditions of 1 cycle at 94°C for 2 min, 30 cycles at 94°C for 20 s, 59°C for 30 s, 72°C for 1 min and 1 cycle at 72°C for 7 min. Lane 1: Marker; lane 2: DNA synthesized using dNTPs; lane 3: DNA synthesized using B-TTP and the other three dNTPs. Primer 1: 5′-CGCCGCCCCCGCCGCG-3′ Primer 2: 5′-CGGCGGCCCGCGGGCG; Template DNA: 5′-CGCCGCCCCCGCCGCG-40N-CGCCCGCGGGCCGCCG-3′.
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Figure 5: Primer extension using the full-length DNA and boronic acid-labeled DNA as templates. Each 50 µl reaction was performed with 1.2 µM primers 1 and 2/template, 0.25 mM of each dNTPs, 0.25 mM of labeled-TTP (B-TTP), and 3.5 units of high-fidelity DNA polymerase (Roche, Indianapolis, Ind.) under conditions of 1 cycle at 94°C for 2 min, 30 cycles at 94°C for 20 s, 59°C for 30 s, 72°C for 1 min and 1 cycle at 72°C for 7 min. Lane 1: Marker; lane 2: DNA synthesized using dNTPs; lane 3: DNA synthesized using B-TTP and the other three dNTPs. Primer 1: 5′-CGCCGCCCCCGCCGCG-3′ Primer 2: 5′-CGGCGGCCCGCGGGCG; Template DNA: 5′-CGCCGCCCCCGCCGCG-40N-CGCCCGCGGGCCGCCG-3′.

Mentions: In order to confirm the general feasibility of incorporating the boronic acid-labeled TTP (B-TTP) into DNA, we have also carried out similar studies using a different template. The results again demonstrated the synthesis of the full-length DNA using B-TTP. Furthermore, using an agarose gel run for a longer time, the boronic acid-labeled DNA was differentiated from the natural DNA that is consistent with the increased molecular weight of the boronic acid-labeled DNA (Figure 5). It should be noted that the boronic acid has a pKa of about 9 (46) and is mostly charge neutral under the electrophoresis conditions (buffer pH 8.3).Figure 5.


Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA.

Lin N, Yan J, Huang Z, Altier C, Li M, Carrasco N, Suyemoto M, Johnston L, Wang S, Wang Q, Fang H, Caton-Williams J, Wang B - Nucleic Acids Res. (2007)

Primer extension using the full-length DNA and boronic acid-labeled DNA as templates. Each 50 µl reaction was performed with 1.2 µM primers 1 and 2/template, 0.25 mM of each dNTPs, 0.25 mM of labeled-TTP (B-TTP), and 3.5 units of high-fidelity DNA polymerase (Roche, Indianapolis, Ind.) under conditions of 1 cycle at 94°C for 2 min, 30 cycles at 94°C for 20 s, 59°C for 30 s, 72°C for 1 min and 1 cycle at 72°C for 7 min. Lane 1: Marker; lane 2: DNA synthesized using dNTPs; lane 3: DNA synthesized using B-TTP and the other three dNTPs. Primer 1: 5′-CGCCGCCCCCGCCGCG-3′ Primer 2: 5′-CGGCGGCCCGCGGGCG; Template DNA: 5′-CGCCGCCCCCGCCGCG-40N-CGCCCGCGGGCCGCCG-3′.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1851626&req=5

Figure 5: Primer extension using the full-length DNA and boronic acid-labeled DNA as templates. Each 50 µl reaction was performed with 1.2 µM primers 1 and 2/template, 0.25 mM of each dNTPs, 0.25 mM of labeled-TTP (B-TTP), and 3.5 units of high-fidelity DNA polymerase (Roche, Indianapolis, Ind.) under conditions of 1 cycle at 94°C for 2 min, 30 cycles at 94°C for 20 s, 59°C for 30 s, 72°C for 1 min and 1 cycle at 72°C for 7 min. Lane 1: Marker; lane 2: DNA synthesized using dNTPs; lane 3: DNA synthesized using B-TTP and the other three dNTPs. Primer 1: 5′-CGCCGCCCCCGCCGCG-3′ Primer 2: 5′-CGGCGGCCCGCGGGCG; Template DNA: 5′-CGCCGCCCCCGCCGCG-40N-CGCCCGCGGGCCGCCG-3′.
Mentions: In order to confirm the general feasibility of incorporating the boronic acid-labeled TTP (B-TTP) into DNA, we have also carried out similar studies using a different template. The results again demonstrated the synthesis of the full-length DNA using B-TTP. Furthermore, using an agarose gel run for a longer time, the boronic acid-labeled DNA was differentiated from the natural DNA that is consistent with the increased molecular weight of the boronic acid-labeled DNA (Figure 5). It should be noted that the boronic acid has a pKa of about 9 (46) and is mostly charge neutral under the electrophoresis conditions (buffer pH 8.3).Figure 5.

Bottom Line: We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization.We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA.DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Center for Biotechnology and Drug Design, Campus Box 4098, Georgia State University, Atlanta, GA 30302-4098, USA.

ABSTRACT
The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.

Show MeSH
Related in: MedlinePlus