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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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The MALDI-TOF mass spectrometric analysis of primer extension products on a 21-nt template using TTP (top, showing a mixture of template and TTP product) and B-TTP (bottom: showing a mixture of template and B-TTP product). The mass difference of 418.6 reflects the incorporation of the boronic acid-labeled thymidine moiety.
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Figure 1: The MALDI-TOF mass spectrometric analysis of primer extension products on a 21-nt template using TTP (top, showing a mixture of template and TTP product) and B-TTP (bottom: showing a mixture of template and B-TTP product). The mass difference of 418.6 reflects the incorporation of the boronic acid-labeled thymidine moiety.

Mentions: Since the goal is the incorporation of the boronic acid moiety into DNA, primer extension using B-TTP (12) was conducted using a short sequence of 21-nt template (5′-GGTTCCACCAGCAACCCGCTA-3′, molecular weight = 6336.2 Da) as the template and a 14-nt primer (5′-TAGCGGGTTGCTGG-3′, molecular weight = 4350.8 Da). The primer extension reaction using natural TTP yielded a DNA product with molecular weight of about 6512 a as determined using MALDI mass spectrometry (calculated molecular weight: 6518.2 a) (Figure 1). The same reaction using B-TTP yielded a DNA product with a molecular weight of about 6941 Da (calculated molecular weight: 6930.2 Da). Such results clearly demonstrated the successful incorporation of the boronic acid-labeled thymidine unit.Figure 1.


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)

The MALDI-TOF mass spectrometric analysis of primer extension products on a 21-nt template using TTP (top, showing a mixture of template and TTP product) and B-TTP (bottom: showing a mixture of template and B-TTP product). The mass difference of 418.6 reflects the incorporation of the boronic acid-labeled thymidine moiety.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 1: The MALDI-TOF mass spectrometric analysis of primer extension products on a 21-nt template using TTP (top, showing a mixture of template and TTP product) and B-TTP (bottom: showing a mixture of template and B-TTP product). The mass difference of 418.6 reflects the incorporation of the boronic acid-labeled thymidine moiety.
Mentions: Since the goal is the incorporation of the boronic acid moiety into DNA, primer extension using B-TTP (12) was conducted using a short sequence of 21-nt template (5′-GGTTCCACCAGCAACCCGCTA-3′, molecular weight = 6336.2 Da) as the template and a 14-nt primer (5′-TAGCGGGTTGCTGG-3′, molecular weight = 4350.8 Da). The primer extension reaction using natural TTP yielded a DNA product with molecular weight of about 6512 a as determined using MALDI mass spectrometry (calculated molecular weight: 6518.2 a) (Figure 1). The same reaction using B-TTP yielded a DNA product with a molecular weight of about 6941 Da (calculated molecular weight: 6930.2 Da). Such results clearly demonstrated the successful incorporation of the boronic acid-labeled thymidine unit.Figure 1.

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