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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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(a) Time-dependent primer extension using B-TTP and TTP: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. Electrophoresis was conducted on 19% acrylamide gel. (b) Primer extension using B-TTP analyzed on 15% acrylamide gel: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. From left to right, lane 1 (from left): M-TTP-DNA; lane 2: co-spot of M-TTP-DNA and TTP-DNA; lane 3: TTP-DNA; lane 4: co-spot of B-TTP-DNA, and TTP-DNA; lane 5 B-TTP-DNA, lane 6: primer.
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Figure 2: (a) Time-dependent primer extension using B-TTP and TTP: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. Electrophoresis was conducted on 19% acrylamide gel. (b) Primer extension using B-TTP analyzed on 15% acrylamide gel: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. From left to right, lane 1 (from left): M-TTP-DNA; lane 2: co-spot of M-TTP-DNA and TTP-DNA; lane 3: TTP-DNA; lane 4: co-spot of B-TTP-DNA, and TTP-DNA; lane 5 B-TTP-DNA, lane 6: primer.

Mentions: More detailed examination of the B-TTP incorporation was conducted through the extension of a 21-nt primer on a 55-nt template. This longer template has three As in the sequence allowing for the incorporation of three Ts or labeled Ts. We first studied the time-dependent incorporation of B-TTP compared with natural TTP using a 32P-labeled primer. Gel electrophoresis results showed that the full-length DNA was obtained from primer extension reactions (Figure 2a) that was confirmed by mass spectrometry. Furthermore, there was no noticeable difference in the rate of incorporation of natural TTP and B-TTP (12). For example, at 0.5 min, neither the B-TTP nor the natural TTP was incorporated significantly. From time 0 to 15 min, there was time-dependent incorporation in both cases. At 15 min, both reactions reached maximal incorporation. Control reactions with only the primer, without enzyme and without added TTP or labeled TTP showed no full-length DNA formation. (The smear in the third lane without TTP was from mismatch pairing and incomplete reaction.) All these indicate that the boronic acid-labeled base, B-TTP (12), was recognized by the Klenow fragment at approximately the same level as natural TTP. It is interesting to note that the B-TTP DNA and TTP-DNA were not well separated when using 19% acrylamide gel (Figure 2a), but were well resolved when using 15% acrylamide gel (Figure 2b).Figure 2.


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)

(a) Time-dependent primer extension using B-TTP and TTP: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. Electrophoresis was conducted on 19% acrylamide gel. (b) Primer extension using B-TTP analyzed on 15% acrylamide gel: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. From left to right, lane 1 (from left): M-TTP-DNA; lane 2: co-spot of M-TTP-DNA and TTP-DNA; lane 3: TTP-DNA; lane 4: co-spot of B-TTP-DNA, and TTP-DNA; lane 5 B-TTP-DNA, lane 6: primer.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 2: (a) Time-dependent primer extension using B-TTP and TTP: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. Electrophoresis was conducted on 19% acrylamide gel. (b) Primer extension using B-TTP analyzed on 15% acrylamide gel: reaction was performed with 5 µM 5′5′-32P-primer/template, 0.4 mM of each dNTPs, 0.4 mM of B-TTP, and Klenow 0.04 units. From left to right, lane 1 (from left): M-TTP-DNA; lane 2: co-spot of M-TTP-DNA and TTP-DNA; lane 3: TTP-DNA; lane 4: co-spot of B-TTP-DNA, and TTP-DNA; lane 5 B-TTP-DNA, lane 6: primer.
Mentions: More detailed examination of the B-TTP incorporation was conducted through the extension of a 21-nt primer on a 55-nt template. This longer template has three As in the sequence allowing for the incorporation of three Ts or labeled Ts. We first studied the time-dependent incorporation of B-TTP compared with natural TTP using a 32P-labeled primer. Gel electrophoresis results showed that the full-length DNA was obtained from primer extension reactions (Figure 2a) that was confirmed by mass spectrometry. Furthermore, there was no noticeable difference in the rate of incorporation of natural TTP and B-TTP (12). For example, at 0.5 min, neither the B-TTP nor the natural TTP was incorporated significantly. From time 0 to 15 min, there was time-dependent incorporation in both cases. At 15 min, both reactions reached maximal incorporation. Control reactions with only the primer, without enzyme and without added TTP or labeled TTP showed no full-length DNA formation. (The smear in the third lane without TTP was from mismatch pairing and incomplete reaction.) All these indicate that the boronic acid-labeled base, B-TTP (12), was recognized by the Klenow fragment at approximately the same level as natural TTP. It is interesting to note that the B-TTP DNA and TTP-DNA were not well separated when using 19% acrylamide gel (Figure 2a), but were well resolved when using 15% acrylamide gel (Figure 2b).Figure 2.

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