8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties

Abstract

We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2′-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson–Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.

Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

“…The emission of 8vdA was shown to be responsive to changes in temperature and solvent, while insensitive to pH changes (between 5–10), displaying desirable properties as a probe (see Section 6.7). Incorporation into oligonucleotides showed sequence dependent, albeit minimal, disruption to duplex stability 567. Much like 2-AP, the intense emission of 8vdA is quenched upon incorporation into oligonucleotides, albeit to a lesser extent 567…”

“…Substituting the hydrogen at the 8 position of adenine with a vinyl moiety results in remarkable photophysical changes compared to the parent nucleobase 567,568. Upon excitation of 8-vinyl-2′-deoxyadenosine (8vdA, 6.129 ) at its absorption maximum (290 nm), an intense emission is observed at 382 nm.…”

Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding

“…Finally, in addition to halogenation of uridines and cytosines for X-ray crystallography, other modifications are commonly used in structural studies of nucleic acids: ribose modification for EPR spectroscopy (Macosko et al 1999), 29-fluoro-nucleotides (Kreutz et al 2005(Kreutz et al , 2006 or 5-fluoropyrimidines (Marshall and Smith 1977;Rastinejad et al 1995) for NMR spectroscopy, introduction of various fluorescent bases for fluorescence studies (Qin and Pyle 1999;Yamana et al 1999;Ben Gaied et al 2005), or, more recently, incorporation of selenium for crystal structure determination (Wilds et al 2002;Hobartner and Micura 2004). Our results highlight the importance of checking the possible influence on RNA folding of even subtle nucleic acids modifications.…”

Fluorescent nucleobases as tools for studying DNA and RNA

“…For example, by adding a vinyl group to the 8 position of either adenine (8vA) or guanine (8vG), the emission can redshift to 382 nm or 400 nm, and result in remarkable quantum yields of 0.66 and 0.72, respectively (Fig. 3e) 44,45 . Another important modification is the addition of external non-emissive aromatic structures to the natural nucleobases.…”