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Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase.

Jarchow-Choy SK, Krueger AT, Liu H, Gao J, Kool ET - Nucleic Acids Res. (2010)

Bottom Line: Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support.For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed.For (dxC)(n), fluorescence enhancement was observed in the polymer.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

ABSTRACT
Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.

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Spectra of xDNTP monomers and of TdT reaction products partially purified by size exclusion. (A) Normalized emission spectra of TdT reaction products with dxATP (green) and monomer dxATP (purple) in buffer (excitation 333 nm). (B) Normalized emission spectrum of the TdT reaction product with dxCTP (blue) and monomer dxCTP (red) in PIPES buffer (excitation 330 nm). Spectra were measured in PIPES buffer (100 mM NaCl, 10 mM MgCl2 and 10 mM Na•PIPES, pH 7.0).
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Figure 4: Spectra of xDNTP monomers and of TdT reaction products partially purified by size exclusion. (A) Normalized emission spectra of TdT reaction products with dxATP (green) and monomer dxATP (purple) in buffer (excitation 333 nm). (B) Normalized emission spectrum of the TdT reaction product with dxCTP (blue) and monomer dxCTP (red) in PIPES buffer (excitation 330 nm). Spectra were measured in PIPES buffer (100 mM NaCl, 10 mM MgCl2 and 10 mM Na•PIPES, pH 7.0).

Mentions: After incubation of an oligodeoxynucleotide primer with dxATP and TdT, size-exclusion chromatography was used to remove excess dNTP. Spectra of the remaining oligonucleotide-enriched fraction showed a distinct change in the spectrum relative to the monomer dxATP (Figure 4A). While the monomer showed an emission maximum at ∼382 nm, the purified product strand showed an emission spectrum with peaks typical of the monomer dxATP as well as a distinct long-wavelength peak at ∼488 nm, characteristic of excimer emission by adjacent dxA bases (33).Figure 4.


Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase.

Jarchow-Choy SK, Krueger AT, Liu H, Gao J, Kool ET - Nucleic Acids Res. (2010)

Spectra of xDNTP monomers and of TdT reaction products partially purified by size exclusion. (A) Normalized emission spectra of TdT reaction products with dxATP (green) and monomer dxATP (purple) in buffer (excitation 333 nm). (B) Normalized emission spectrum of the TdT reaction product with dxCTP (blue) and monomer dxCTP (red) in PIPES buffer (excitation 330 nm). Spectra were measured in PIPES buffer (100 mM NaCl, 10 mM MgCl2 and 10 mM Na•PIPES, pH 7.0).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045586&req=5

Figure 4: Spectra of xDNTP monomers and of TdT reaction products partially purified by size exclusion. (A) Normalized emission spectra of TdT reaction products with dxATP (green) and monomer dxATP (purple) in buffer (excitation 333 nm). (B) Normalized emission spectrum of the TdT reaction product with dxCTP (blue) and monomer dxCTP (red) in PIPES buffer (excitation 330 nm). Spectra were measured in PIPES buffer (100 mM NaCl, 10 mM MgCl2 and 10 mM Na•PIPES, pH 7.0).
Mentions: After incubation of an oligodeoxynucleotide primer with dxATP and TdT, size-exclusion chromatography was used to remove excess dNTP. Spectra of the remaining oligonucleotide-enriched fraction showed a distinct change in the spectrum relative to the monomer dxATP (Figure 4A). While the monomer showed an emission maximum at ∼382 nm, the purified product strand showed an emission spectrum with peaks typical of the monomer dxATP as well as a distinct long-wavelength peak at ∼488 nm, characteristic of excimer emission by adjacent dxA bases (33).Figure 4.

Bottom Line: Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support.For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed.For (dxC)(n), fluorescence enhancement was observed in the polymer.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

ABSTRACT
Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.

Show MeSH
Related in: MedlinePlus