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Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates.

Guerniou V, Gillet R, Berrée F, Carboni B, Felden B - Nucleic Acids Res. (2007)

Bottom Line: All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro.The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide.These new conjugates are now being tested for activity on infected hepatic cell lines.

View Article: PubMed Central - PubMed

Affiliation: Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France.

ABSTRACT
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

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Related in: MedlinePlus

Chemical procedure used to couple the functional groups to the 5′ or to the 3′-ends of the DNA oligonucleotide. The activated ester was added to the amino-oligonucleotide (DNA or 2′-O-Me-RNA oligonucleotide) to form a peptide bond. The imidazole-conjugated oligonucleotides were obtained from dihydrourocanic acid. The DNA amino-oligonucleotide was coupled either at the 5′ or the 3′-end, they are respectively denoted 5′-imidazole (5′-Im, 1) and 3′-imidazole (3′-Im, 4). The imidazole-coupled 2′-O-Me-RNA oligonucleotide, denoted 5′-imidazole_RNA (5′-Im-RNA, 5), was coupled at the 5′-end. Urocanic acid and benzimidazoleacetic acid functional groups were also coupled at the 5′-end of the DNA oligonucleotide. The corresponding oligonucleotides are respectively denoted 5′-UA (2) and 5′-BI (3).
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Figure 2: Chemical procedure used to couple the functional groups to the 5′ or to the 3′-ends of the DNA oligonucleotide. The activated ester was added to the amino-oligonucleotide (DNA or 2′-O-Me-RNA oligonucleotide) to form a peptide bond. The imidazole-conjugated oligonucleotides were obtained from dihydrourocanic acid. The DNA amino-oligonucleotide was coupled either at the 5′ or the 3′-end, they are respectively denoted 5′-imidazole (5′-Im, 1) and 3′-imidazole (3′-Im, 4). The imidazole-coupled 2′-O-Me-RNA oligonucleotide, denoted 5′-imidazole_RNA (5′-Im-RNA, 5), was coupled at the 5′-end. Urocanic acid and benzimidazoleacetic acid functional groups were also coupled at the 5′-end of the DNA oligonucleotide. The corresponding oligonucleotides are respectively denoted 5′-UA (2) and 5′-BI (3).

Mentions: The procedure for coupling the amino-oligonucleotides (Figure 2) was adapted from (37). 30 μmol (1 eq) of acid RCO2H were activated during 150 min under argon with 1.2 eq of PyBOP, 1.5 eq of HOBT and 3 eq of freshly dried N-methylmorpholine in 1 ml of dry DMF. Fifty micro liters of this solution were added to 40 nmol of the amino-oligonucleotide in 150 μl of buffer (133 mM NaHCO3/Na2CO3, pH 9). One hour later, 50 μl of the activated ester were added to the oligonucleotide solution. This addition was repeated four times. The mixture was kept overnight at room temperature. Prior to analysis and purification, the oligonucleotides were precipitated with 0.1 vol of 3 M sodium acetate (pH 5.3) and six volumes of absolute ethanol. The pellets were dissolved in 100 μl of water. DNA concentration was assessed by UV measurement at 260 nm. The coupling protocol was the same for the DNA amino-oligonucleotide and its 2′-O-Me-RNA analogue.Figure 2.


Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates.

Guerniou V, Gillet R, Berrée F, Carboni B, Felden B - Nucleic Acids Res. (2007)

Chemical procedure used to couple the functional groups to the 5′ or to the 3′-ends of the DNA oligonucleotide. The activated ester was added to the amino-oligonucleotide (DNA or 2′-O-Me-RNA oligonucleotide) to form a peptide bond. The imidazole-conjugated oligonucleotides were obtained from dihydrourocanic acid. The DNA amino-oligonucleotide was coupled either at the 5′ or the 3′-end, they are respectively denoted 5′-imidazole (5′-Im, 1) and 3′-imidazole (3′-Im, 4). The imidazole-coupled 2′-O-Me-RNA oligonucleotide, denoted 5′-imidazole_RNA (5′-Im-RNA, 5), was coupled at the 5′-end. Urocanic acid and benzimidazoleacetic acid functional groups were also coupled at the 5′-end of the DNA oligonucleotide. The corresponding oligonucleotides are respectively denoted 5′-UA (2) and 5′-BI (3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175329&req=5

Figure 2: Chemical procedure used to couple the functional groups to the 5′ or to the 3′-ends of the DNA oligonucleotide. The activated ester was added to the amino-oligonucleotide (DNA or 2′-O-Me-RNA oligonucleotide) to form a peptide bond. The imidazole-conjugated oligonucleotides were obtained from dihydrourocanic acid. The DNA amino-oligonucleotide was coupled either at the 5′ or the 3′-end, they are respectively denoted 5′-imidazole (5′-Im, 1) and 3′-imidazole (3′-Im, 4). The imidazole-coupled 2′-O-Me-RNA oligonucleotide, denoted 5′-imidazole_RNA (5′-Im-RNA, 5), was coupled at the 5′-end. Urocanic acid and benzimidazoleacetic acid functional groups were also coupled at the 5′-end of the DNA oligonucleotide. The corresponding oligonucleotides are respectively denoted 5′-UA (2) and 5′-BI (3).
Mentions: The procedure for coupling the amino-oligonucleotides (Figure 2) was adapted from (37). 30 μmol (1 eq) of acid RCO2H were activated during 150 min under argon with 1.2 eq of PyBOP, 1.5 eq of HOBT and 3 eq of freshly dried N-methylmorpholine in 1 ml of dry DMF. Fifty micro liters of this solution were added to 40 nmol of the amino-oligonucleotide in 150 μl of buffer (133 mM NaHCO3/Na2CO3, pH 9). One hour later, 50 μl of the activated ester were added to the oligonucleotide solution. This addition was repeated four times. The mixture was kept overnight at room temperature. Prior to analysis and purification, the oligonucleotides were precipitated with 0.1 vol of 3 M sodium acetate (pH 5.3) and six volumes of absolute ethanol. The pellets were dissolved in 100 μl of water. DNA concentration was assessed by UV measurement at 260 nm. The coupling protocol was the same for the DNA amino-oligonucleotide and its 2′-O-Me-RNA analogue.Figure 2.

Bottom Line: All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro.The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide.These new conjugates are now being tested for activity on infected hepatic cell lines.

View Article: PubMed Central - PubMed

Affiliation: Biochimie Pharmaceutique, Inserm U835, Upres JE 2311, Université de Rennes 1, France.

ABSTRACT
Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the 'cap-independent' translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5' or 3'-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37 degrees C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the 'IRES-dependent' translation in vitro. The 5'-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

Show MeSH
Related in: MedlinePlus