Tautomers of a Fluorescent G Surrogate and Their Distinct Photophysics Provide Additional Information Channels

Abstract

Thienoguanosine ( th G) is an isomorphic nucleoside analogue acting as a faithful fluorescent substitute of G, with respectable quantum yield in oligonucleotides. Photophysical analysis of th G reveals the existence of two ground-state tautomers with significantly shifted absorption and emission wavelengths, and high quantum yield in buffer. Using (TD)-DFT calculations, the tautomers were identified as the H1 and H3 keto-amino tautomers. When incorporated into the loop of (−)PBS, the (−)DNA copy of the HIV-1 primer binding site, both tautomers are observed and show differential sensitivity to protein binding. The red-shifted H1 tautomer is strongly favored in matched (−)/(+)PBS duplexes, while the relative emission of the H3 tautomer can be used to detect single nucleotide polymorphisms. These tautomers and their distinct environmental sensitivity provide unprecedented information channels for analyzing G residues in oligonucleotides and their complexes. Keywordsab initio calculations; fluorescence; molecular modeling; nucleic acids; tautomerismThe structure, acid-base features, and tautomeric equilibria of the canonical and noncanonical nucleobases found in nucleic acids have been the subject of intense investigation for decades. [1] While the role of minor tautomers in mutagenesis has been one of the primary foci, [2] recent observations suggest that such isomeric nucleobases also play key roles in regular nucleic acid structure and function. [3] As the population of distinct tautomeric forms is impacted by their micro-environment, this added level of complexity also provides opportunities to further advance our understanding of nucleic acid structure and dynamics.In this context, emissive nucleoside analogues, which have become powerful biophysical tools, [4] provide unique prospects. A tautomerizable nucleoside analogue, where the tautomers would have distinct absorption and emission spectra, could be instrumental for [5] and identify two environmentally sensitive ground-state tautomeric forms ( Figure 1) which display distinct absorption and emission spectra. The equilibrium between the two tautomers is mainly governed by the hydrogen-bond donor properties of the solvent. Their observed sensitivity to the microenvironment was rationalized by ab initio calculations. By exploring single-and double-stranded th Gcontaining oligonucleotides, as well as DNA-protein complexes, we illustrate that this probe provides compelling biophysical information and greater insight compared to monochromatic or ratiometric fluorescent nucleosides.The emission spectra of either th G or d th G in water and methanol are surprisingly complex. [5a,b] Excitation at both λ=360 and 380 nm gives a similar emission spectrum (Figure 2a, orange) centered at λ=468 nm. When the excitation energy is progressively increased, a blue-shifted emission with a maximum at λ=400 nm appears and becomes dominant for excitation below λ=300 nm ( Figure 2a, magenta and blue). The simplest interpretation is that th G exhibits two ground-state ...

Low-Temperature Luminescent Studies of Emissive Guanine Substitute for the Detection of Biopolymers

“…The optical absorption spectrum at room temperature of a d th G solution in buffer (Fig. 2) practically coincides with that reported in [3] and the optical absorption spectrum of the d th G solution in water. Most probably, the wide band with the maximum at 325 nm is a result of the addition of two bands associated with the optical absorption of two tautomers, respectively.…”

“…The long-wave bands of these fluorescence excitation spectra possess two unsharp maxima at 315 and 334 nm. This fact confirms the deconvolution of the absorption spectrum of d th G obtained in [3] in two ground-state tautomers even at a temperature of 78 K. The same situation is for the aqueous solutions of d th G.…”

“…In buffer solutions, the emissions of both tautomers are equal in contrast to aqueous solutions. This fact could be explained by that the equilibrium between two tautomers, as was shown in [3], is governed by the Hbond donor properties of the solvent. The redistri-bution of fluorescence emission intensities in aqueous solutions could be a result of the singlet excitation energy transfer (possibly, direct and reverse ones depending on pH) between these two tautomers.…”

“…Two ground-state keto-enol tautomers of d th G early reported in [3] with shifted emission spectra that displayed distinct absorption and emission spectra were identified. The equilibrium between these two tautomers is mainly governed by the H-bond donor properties of the solvent.…”

“…At room temperature, the quantum yield of nucleotides is reported to be low, about 10 −5 -10 −4 [1]. The detection of nucleic acids becomes possible using luminescent probes ("modified" nucleosides, tautomerizable nucleoside analogs) with a respectable quantum yield of luminescence at room temperature which could be incorporated in the biopolymer macromolecule chain [2,3]. These probes would have distinct absorption and emission spectra and, due to their specific energy structure, could be additional traps of singlet and triplet excitations in the biopolymers of nucleic acids.…”