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.

DNA-Multichromophore Systems

“…87 More recently, the TdT enzyme has also been used to incorporate benzo-expanded deoxynucleoside analogs, dxATP and dxCTP, forming fluorescent homopolymers. 263 …”

DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides

“…Size-expanded dxNTPs, which contain a benzene ring within the base moiety of each nucleoside (Figure 1A), were synthesized as described (19). These expanded nucleosides form canonical base pairs and increase the width of double-stranded DNA by 2.4 Å (14).…”

“…The large nucleobase is likely to induce steric hindrance within the active site of DNA polymerases, which may prevent proper positioning of the nucleotide near the 3′-hydroxyl at the primer terminus for the phosphodiester transfer reaction. Interestingly, previous studies have shown efficient utilization of dxNMPs by terminal deoxynucleotidyl transferase, which unlike common DNA polymerases does not require a template for the nucleotidyl transfer, thus potentially explaining its ability to accommodate large unnatural nucleotides (19). …”

Fluorescent DNA-based enzyme sensors

“…133 (A, B) Spectra of xDNTP monomers (purple, red) and of TdT reaction products (green, blue). (A) Incorporation of dxATP; (B) incorporation of dxCTP.…”

“…(C) dxATP without TdT; (D) dxATP with TdT; (E) dxCTP without TdT; (F) dxCTP with TdT. 133 Reprinted from ref. 133 by permission of Oxford University Press, Copyright 2010.…”