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

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.

Figure 1.

Expanded DNA nucleoside triphosphates used in this study.

Scheme 1.

Synthesis of CPG solid support with primer attached. (a) dimethoxytrityl chloride, N, N-diisopropylethylamine, 4-dimethylaminopyridine, dichloromethane, 2 h, room temparature, 92% (b) LiOH-H₂O, THF/MeOH/H₂O (3:1:1), 24 h, room temparature, 59% (c) HATU (peptide coupling agent), 4-dimethylaminopyridine, N, N-diisopropylethylamine, diemethylformamide, room temparature, 75%.

Scheme 2.

Addition of dxATP and dxCTP to (dT)25 primer attached to CPG bead.

Figure 2.

Autoradiogram of a polyacrylamide gel showing TdT insertion of xDNA and DNA nucleoside triphosphates after 1 h. Lane 1: incorporation of dTTP, 10 µM; lane 2: dGTP, 10 µM; lane 3: dCTP, 10 µM; lane 4: dATP, 10 µM; lane 5: dxTTP, 10 µM; lane 6: dxGTP, 10 µM; lane 7: dxCTP, 10 µM; lane 8: dxATP, 10 µM. M: 10 bp size marker; P: unreacted DNA primer (sequence 5′-dATACCAAAGT-3′).

Figure 3.

Steady-state incorporation efficiencies (as V_max/K_m) for the single nucleotide incorporation of expanded and natural nucleoside triphosphates into a primer by TdT. Data were obtained by gel electrophoretic analysis using radiolabeled primers.

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).

Figure 5.

Changes in fluorescence emission during TdT-mediated polymerization of xDNA. (A) dxATP incorporation measured at 385 nm. Reaction aliquots measured in PIPES buffer (100 mM NaCl, 10 mM MgCl₂ and 10 mM Na•PIPES, pH 7.0, excitation 333 nm). (B) dxCTP incorporation measured at 395 nm (excitation 330 nm). Curves are exponential fits to the data.

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).