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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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All the DNA conjugates bind to the HCV IRES at the predicted location within the RNA sequence. (a) Representative native gel retardation assay between the radiolabelled 3′-imidazole conjugate and increasing concentrations (from 0 to 2000 nM) of purified unlabelled IRES RNA. The slower migrating band corresponding to the IRES-conjugate duplex is indicated by a star. Apparent dissociation constants between each conjugate and the IRES were calculated from three independent experiments. (b) RNase H cleavage assays between each of the unlabelled conjugates and labelled IRES RNA. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions demonstrates that the complex between each of the antisense oligonucleotides and the IRES takes place at the predicted location (indicated by the arrow and the black circle) deduced by nucleotide pairing, at hairpin IIId.
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Figure 3: All the DNA conjugates bind to the HCV IRES at the predicted location within the RNA sequence. (a) Representative native gel retardation assay between the radiolabelled 3′-imidazole conjugate and increasing concentrations (from 0 to 2000 nM) of purified unlabelled IRES RNA. The slower migrating band corresponding to the IRES-conjugate duplex is indicated by a star. Apparent dissociation constants between each conjugate and the IRES were calculated from three independent experiments. (b) RNase H cleavage assays between each of the unlabelled conjugates and labelled IRES RNA. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions demonstrates that the complex between each of the antisense oligonucleotides and the IRES takes place at the predicted location (indicated by the arrow and the black circle) deduced by nucleotide pairing, at hairpin IIId.

Mentions: The binding affinities of the four labelled conjugates for increasing amounts of the complete IRES from HCV were assayed by native gel retardation assays and compared to that of the naked oligonucleotide sequence (Figure 3a). A retarded band (Figure 3a, star), not detected in the control lane in the absence of the RNA, corresponds to the RNA–conjugate complex. Two additional negative controls were performed in the same conditions. First, we incubated the HCV IRES with a non-specific oligonucleotide (NS). This mismatched sequence does not bind the IRES RNA, up to the higher concentration used for the antisense oligonucleotide (data not shown). Then, the ECMV IRES was incubated with the antisense (AS). No retarded bands were detected (data not shown). The apparent dissociation constants (Kd) of the HCV IRES-oligonucleotide conjugates complex were calculated. The uncoupled antisense oligonucleotide has a ∼70 nM apparent Kd. Adding any of the three imidazole groups to the 3′ or 5′ end of the antisense oligonucleotide does not modify significantly this binding affinity (data not shown).Figure 3.


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)

All the DNA conjugates bind to the HCV IRES at the predicted location within the RNA sequence. (a) Representative native gel retardation assay between the radiolabelled 3′-imidazole conjugate and increasing concentrations (from 0 to 2000 nM) of purified unlabelled IRES RNA. The slower migrating band corresponding to the IRES-conjugate duplex is indicated by a star. Apparent dissociation constants between each conjugate and the IRES were calculated from three independent experiments. (b) RNase H cleavage assays between each of the unlabelled conjugates and labelled IRES RNA. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions demonstrates that the complex between each of the antisense oligonucleotides and the IRES takes place at the predicted location (indicated by the arrow and the black circle) deduced by nucleotide pairing, at hairpin IIId.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: All the DNA conjugates bind to the HCV IRES at the predicted location within the RNA sequence. (a) Representative native gel retardation assay between the radiolabelled 3′-imidazole conjugate and increasing concentrations (from 0 to 2000 nM) of purified unlabelled IRES RNA. The slower migrating band corresponding to the IRES-conjugate duplex is indicated by a star. Apparent dissociation constants between each conjugate and the IRES were calculated from three independent experiments. (b) RNase H cleavage assays between each of the unlabelled conjugates and labelled IRES RNA. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions demonstrates that the complex between each of the antisense oligonucleotides and the IRES takes place at the predicted location (indicated by the arrow and the black circle) deduced by nucleotide pairing, at hairpin IIId.
Mentions: The binding affinities of the four labelled conjugates for increasing amounts of the complete IRES from HCV were assayed by native gel retardation assays and compared to that of the naked oligonucleotide sequence (Figure 3a). A retarded band (Figure 3a, star), not detected in the control lane in the absence of the RNA, corresponds to the RNA–conjugate complex. Two additional negative controls were performed in the same conditions. First, we incubated the HCV IRES with a non-specific oligonucleotide (NS). This mismatched sequence does not bind the IRES RNA, up to the higher concentration used for the antisense oligonucleotide (data not shown). Then, the ECMV IRES was incubated with the antisense (AS). No retarded bands were detected (data not shown). The apparent dissociation constants (Kd) of the HCV IRES-oligonucleotide conjugates complex were calculated. The uncoupled antisense oligonucleotide has a ∼70 nM apparent Kd. Adding any of the three imidazole groups to the 3′ or 5′ end of the antisense oligonucleotide does not modify significantly this binding affinity (data not shown).Figure 3.

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