Fluorescent Nucleoside Analogues Research Articles

Probing DNA sequences in solution with a monomer-excimer fluorescence color change

The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambdamax= 381 398 nm) to green-white excimer emission (lambdamax= 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.

Study of the substrate-binding properties of bovine liver adenosine kinase and inhibition by fluorescent nucleoside analogues.

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine to AMP with ATP as phosphate donor. Intrinsic fluorescence of bovine liver AK was shown previously to be a sensitive probe to quantify the binding of substrates to the enzyme [Elaloui, A., Divita, G., Maury, G., Imbach, J.-L. & Goody, R. S. (1994) Eur. J Biochem. 221, 839-846]. AK contains two catalytic, sites: a high-affinity site, which binds adenosine and AMP selectively; and a site for ATP and ADP. In the present work, these two sites were characterized by combining the quenching of protein fluorescence induced by the binding of the ligands and the fluorescence enhancement observed upon binding of the N-methylanthraniloyl-derivated nucleotides or adenosine. A new fluorescent analog of adenosine, 5'-N-methylanthraniloyl-adenosine, was synthesized and shown to bind selectively to the high-affinity adenosine-binding site with an affinity similar to that of adenosine (Kd 1 microM). In contrast, 2'(3')-N-methylanthraniloyl derivatives of ATP, adenosine (5')tetraphospho(5')adenosine (Ap4A), and adenosine (5')pentaphospho(5')adenosine (Ap5A), bind to the enzyme at the ATP site. Methylantraniloyl derivatives of ATP and adenosine were used as tools for selective characterization of a series of adenosine analogues. The bisubstrate inhibitors Ap4A and Ap5A bind to the ATP site with high affinity and apparently not to the adenosine site, thus acting more as ATP analogues than true bisubstrate ligands. The binding properties of a series of adenosine analogues were strongly dependent on the structural modifications on adenosine. The analogues modified at positions 2' or 3' show similar affinities for AK as that of adenosine, whereas adenosine analogues modified at the base present a relatively low affinity for the enzyme.

Fluorescence Properties of Pteridine Nucleoside Analogs as Monomers and Incorporated into Oligonucleotides

Eighteen fluorescent pteridine-based nucleoside analogs have been prepared that are suitable for synthesis as phosphoramidites and site-specific incorporation into oligonucleotides. Their quantum yields ranged from ≤0.03 to 0.88. The maximum excitation and emission wavelengths of seven selected probes with quantum yields >0.15 ranged from 334 to 358 and 400 to 444 nm, respectively. Fluorescence decay curves of the seven probes were biexponential, and the mean intensity-weighted lifetimes ranged from 0.87 to 6.54 ns. Incorporation of probes 4 and 17 (3-methylisoxanthopterin and 6-methylisoxanthopterin) into oligonucleotides significantly quenched their fluorescence signal, and the degree of quench correlated with the number and proximity of purines in the oligonucleotide. Incorporation also resulted in a shift in absorbance-, emission-, and decay-associated spectra for 6-methylisoxanthopterin. An increase in the complexity of the decay curve and a decrease in the mean lifetime occurred for both probes. Formation of double-stranded oligonucleotides did not substantially increase the degree of quenching but generally increased the complexity of decay curves and decreased the mean lifetimes. Melting temperature,Tm, depression equivalent to that of a single base pair mismatch was observed in 3-methylisoxanthopterin-containing double-stranded oligonucleotides, while theTmof 6-methylisoxanthopterin-containing double-stranded oligonucleotides were unperturbed, e.g., equivalent to unlabeled double-stranded oligonucleotides. This new class of fluorophore yields promising probes for the study of protein/DNA interactions.

Fluorescent nucleoside analogues : Synthesis of fluorescent pyrido[2,1-i]purines, and their corresponding ribosides

Fluorescent pyrido[2,1-i]purines can in principle be obtained via Michael-addition of a suitable anion of a purine derivative to an acetylenic ester, followed by based-catalyzed cyclization, as depicted in Scheme II. 6-Substituted purine-derivatives are obtained via nucleophilic substitution of 6-chloro- and 6-methylsulfonylpurine ( 8a and 8b ). In the presence of methyl propiolate and sodium methoxide, before cyclization, two consecutive Michael-additions take place, leading to 13 and 14 . With substituted acetylenic esters, cyclization occurs after one Michael-addition. Michael-additions with ethylenic esters did not lead to expected cyclization products, except in cases where oxidation took place. -For the conversion of the pyrido[2,1-i]purines into the corresponding ribosides protection against nucleophilic attack was necessary.