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

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

Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer

Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (−) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(−)PBS annealing mechanism by activating a loop–loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (−)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(−)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can ‘freeze’ PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA

Etude du rôle de la protéine NCp7 dans le mécanisme d'hybridation de la séquence PBS lors du second saut de brin de la transcription inverse de VIH-1

La protéine NCp7 est une cible potentielle de choix pour une thérapie anti-VIH car elle est impliquée dans de nombreuses étapes du cycle viral, et sa séquence est hautement conservée. En combinant les techniques de spectroscopie de fluorescence et de RMN, nous avons montré que NCp7 modifie la conformation de la boucle de PBS et déstabilise l extrémité supérieure de la tige. De ce fait, NCp7 active l hybridation de PBS en stimulant la formation de complexes boucle-boucle, alors qu en absence de protéine l extrémité protrudente constitue le site principal de nucléation. Les modifications de la boucle de PBS induites par NCp7 permettent la formation d homodimères qui jouent probablement un rôle dans la variabilité génétique du virus. Les propriétés spectroscopiques de la 8-vinyl-déoxyadenosine, un analogue fluorescent de l adénine, ont été étudiées. Par ses propriétés, cette sonde apparaît supérieure à la 2-AP.The NCp7 protein is a potential target for an anti-HIV therapy since it is involved in numerous stages of the viral cycle, and its sequence is highly conserved. By combining fluorescence spectroscopy and RMN techniques, we showed that NCp7 modifies the conformation of the PBS loop and destabilizes the upper extremity of the stem. Therefore, NCp7 activates the PBS annealing by stimulating the formation of kissing complexes , while in absence of protein, the protruding extremity constitutes the main nucleation site. The modifications of PBS loop inferred by NCp7 also allow homodimers of PBS, which probably play a role in the genetic variability of the virus. The spectroscopic properties of the 8-vinyl-déoxyadenosine, a fluorescent analogue of the adenine, were studied. By its properties, this probe seems superior to the 2-AP.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Interaction between dietary bioactive peptides of short length and bile salts in submicellar or micellar state

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Chemical synthesis and development of modified xylosides as potential inhibitors targeting β4GalT7, a key enzyme in glycosaminoglycan biosynthesis initiation.

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Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer

10.1093/nar/gkr274NUCLEIC ACIDS RESEARCH39156633-664

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

International audienceThe human immunodeficiency virus type 1 nucleocapsid protein (NCp7) plays an important role in the second strand transfer during reverse transcription. It promotes annealing of the 18-nucleotide complementary DNA primer-binding site (PBS) sequences at the 3' ends of (-)DNA and (+)DNA. NMR studies show that NCp7(12-55) and NCp7(1-55) interact at the 5' end of the loop of DeltaP(-)PBS, a (-)PBS derivative without the 3' protruding sequence, in a slow-exchange equilibrium. This interaction is mediated through the binding of the hydrophobic plateau (Val13, Phe16, Thr24, Ala25, Trp37, and Met46) on the zinc finger domain of both peptides to the 5-CTG-7 sequence of DeltaP(-)PBS. The stacking of the Trp37 aromatic ring with the G7 residue likely constitutes the determinant factor of the interaction. Although NCp7(12-55) does not melt the DeltaP(-)PBS stem-loop structure, it opens the loop and weakens the C5.G11 base pair next to the loop. Moreover, NCp7(12-55) was also found to bind but with lower affinity to the 10-CGG-12 sequence in an intermediate-exchange equilibrium on the NMR time scale. The loop modifications may favour a kissing interaction with the complementary (+)PBS loop. Moreover, the weakening of the upper base pair of the stem likely promotes the melting of the stem that is required to convert the kissing complex into the final (+/-)PBS extended duplex

Synthesis of a library of variously modified 4-methylumbelliferyl xylosides and a structure–activity study of human β4GalT7

International audienceProteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG)chains covalently attached to a core protein through a tetrasaccharide linker. The biosynthesis of PGs iscomplex and involves a large number of glycosyltranferases. Here we present a structure–activity study ofhuman β4GalT7, which transfers the first Gal residue onto a xyloside moiety of the linkage region. Anefficient and regiocontrolled synthesis of a library of modified analogs of 4-methylumbelliferyl xyloside(XylMU) is reported herein. Hydroxyl groups at the position C-2, C-3 or C-4 have been epimerized and/orreplaced by a hydrogen or a fluorine, while the anomeric oxygen was replaced by either a sulfur or asulfone. The effect of these compounds on human β4GalT7 activity in vitro and on GAG biosynthesisin cellulo was then evaluated

A versatile strategy to synthesize N -methyl-anthranilic acid-labelled glycoprobes for fluorescence-based screening assays

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