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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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Fluorescence microscopy images of TdT-mediated xDNA reaction products on CPG with (dT)25 primer attached. (A) [dxATP] = 68 µM, TdT omitted. (B) [dxATP] = 68 µM, [TdT] = 1 U/µl. (C) [dxCTP] = 140 µM, TdT omitted. (D) [dxCTP] = 140 µM, [TdT] = 1 U/µl. Excitation 330–380 nm for all images; emission was measured with a 420 nm long-pass filter for (A), (B) and 400 nm long-pass filter for (C and D).
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Figure 6: Fluorescence microscopy images of TdT-mediated xDNA reaction products on CPG with (dT)25 primer attached. (A) [dxATP] = 68 µM, TdT omitted. (B) [dxATP] = 68 µM, [TdT] = 1 U/µl. (C) [dxCTP] = 140 µM, TdT omitted. (D) [dxCTP] = 140 µM, [TdT] = 1 U/µl. Excitation 330–380 nm for all images; emission was measured with a 420 nm long-pass filter for (A), (B) and 400 nm long-pass filter for (C and D).

Mentions: Images of CPG beads after TdT reactions with these unnatural monomers under an epifluorescence microscope revealed visible fluorescence changes for both dxATP and dxCTP incorporation (Figure 6). Blue/green fluorescence (>420 nm) was observed after TdT was added to a reaction mixture containing the primer-substituted CPG-beads and dxATP (Figure 6A and B). The same procedure was used to image glass substrates following reaction of TdT with the CPG-attached primer and dxCTP. Images measured at >400 nm showed a blue fluorescence enhancement of the beads after addition of TdT to a mixture containing the CPG beads and dxCTP (Figure 6C and D).Figure 6.


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)

Fluorescence microscopy images of TdT-mediated xDNA reaction products on CPG with (dT)25 primer attached. (A) [dxATP] = 68 µM, TdT omitted. (B) [dxATP] = 68 µM, [TdT] = 1 U/µl. (C) [dxCTP] = 140 µM, TdT omitted. (D) [dxCTP] = 140 µM, [TdT] = 1 U/µl. Excitation 330–380 nm for all images; emission was measured with a 420 nm long-pass filter for (A), (B) and 400 nm long-pass filter for (C and D).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Fluorescence microscopy images of TdT-mediated xDNA reaction products on CPG with (dT)25 primer attached. (A) [dxATP] = 68 µM, TdT omitted. (B) [dxATP] = 68 µM, [TdT] = 1 U/µl. (C) [dxCTP] = 140 µM, TdT omitted. (D) [dxCTP] = 140 µM, [TdT] = 1 U/µl. Excitation 330–380 nm for all images; emission was measured with a 420 nm long-pass filter for (A), (B) and 400 nm long-pass filter for (C and D).
Mentions: Images of CPG beads after TdT reactions with these unnatural monomers under an epifluorescence microscope revealed visible fluorescence changes for both dxATP and dxCTP incorporation (Figure 6). Blue/green fluorescence (>420 nm) was observed after TdT was added to a reaction mixture containing the primer-substituted CPG-beads and dxATP (Figure 6A and B). The same procedure was used to image glass substrates following reaction of TdT with the CPG-attached primer and dxCTP. Images measured at >400 nm showed a blue fluorescence enhancement of the beads after addition of TdT to a mixture containing the CPG beads and dxCTP (Figure 6C and D).Figure 6.

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