Limits...
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.

Show MeSH

Related in: MedlinePlus

In vitro cleavage assays between the conjugates and the IRES from HCV. Purified 3′-labelled IRES is incubated with either 50 μM free imidazole or increasing concentrations of the naked or the 3′/5′ imidazole-coupled antisense oligonucleotides for one hour at 37°C. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions as well as partial alkaline RNA hydrolysis allows the mapping of the cleavage sites within the IRES RNA sequence. The arrow points to the cleavage site, between G271 and C272, induced by conjugate 1. The stars point to three additional cleavage sites induced by conjugates 1 and 4, mapped between U297 and A298 in domain IIIe (*1), between G171 and A172 between domains IIIa and IIIb (*2) and between G181 and A182 in domain IIIb (*3). Note that the two highest concentrations of the naked oligonucleotide elicit a weak cleavage between G171 and A172 (*2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175329&req=5

Figure 4: In vitro cleavage assays between the conjugates and the IRES from HCV. Purified 3′-labelled IRES is incubated with either 50 μM free imidazole or increasing concentrations of the naked or the 3′/5′ imidazole-coupled antisense oligonucleotides for one hour at 37°C. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions as well as partial alkaline RNA hydrolysis allows the mapping of the cleavage sites within the IRES RNA sequence. The arrow points to the cleavage site, between G271 and C272, induced by conjugate 1. The stars point to three additional cleavage sites induced by conjugates 1 and 4, mapped between U297 and A298 in domain IIIe (*1), between G171 and A172 between domains IIIa and IIIb (*2) and between G181 and A182 in domain IIIb (*3). Note that the two highest concentrations of the naked oligonucleotide elicit a weak cleavage between G171 and A172 (*2).

Mentions: We designed and performed additional experiments, in the absence of RNase H, in which the refolded IRES RNA (Figure 1) is incubated from 5 to 60 min with increasing concentrations (from 0.5 to 50 μM) of either the naked DNA antisense AS or the oligonucleotides conjugates 1 or 4. As negative controls, the naked antisense and the free, uncoupled imidazole were used at concentrations and incubation times identical to those used for the conjugates (Material and Methods section). Alkaline hydrolysis and RNase T1 and U2 digestions of the RNA allow the positioning of the cleavage sites. One-hour incubation at the lower concentration (0.5 μM) is required to observe a significant cleavage at the expected location (Figure 4, arrow) only for the 5′-coupled imidazole conjugate 1. Three strong additional cleavages are observed for conjugates 1 and 4 (stars in Figure 4). With the naked oligonucleotide AS, these cleavages are very weak but can also be detected.Figure 4.


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)

In vitro cleavage assays between the conjugates and the IRES from HCV. Purified 3′-labelled IRES is incubated with either 50 μM free imidazole or increasing concentrations of the naked or the 3′/5′ imidazole-coupled antisense oligonucleotides for one hour at 37°C. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions as well as partial alkaline RNA hydrolysis allows the mapping of the cleavage sites within the IRES RNA sequence. The arrow points to the cleavage site, between G271 and C272, induced by conjugate 1. The stars point to three additional cleavage sites induced by conjugates 1 and 4, mapped between U297 and A298 in domain IIIe (*1), between G171 and A172 between domains IIIa and IIIb (*2) and between G181 and A182 in domain IIIb (*3). Note that the two highest concentrations of the naked oligonucleotide elicit a weak cleavage between G171 and A172 (*2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: In vitro cleavage assays between the conjugates and the IRES from HCV. Purified 3′-labelled IRES is incubated with either 50 μM free imidazole or increasing concentrations of the naked or the 3′/5′ imidazole-coupled antisense oligonucleotides for one hour at 37°C. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions as well as partial alkaline RNA hydrolysis allows the mapping of the cleavage sites within the IRES RNA sequence. The arrow points to the cleavage site, between G271 and C272, induced by conjugate 1. The stars point to three additional cleavage sites induced by conjugates 1 and 4, mapped between U297 and A298 in domain IIIe (*1), between G171 and A172 between domains IIIa and IIIb (*2) and between G181 and A182 in domain IIIb (*3). Note that the two highest concentrations of the naked oligonucleotide elicit a weak cleavage between G171 and A172 (*2).
Mentions: We designed and performed additional experiments, in the absence of RNase H, in which the refolded IRES RNA (Figure 1) is incubated from 5 to 60 min with increasing concentrations (from 0.5 to 50 μM) of either the naked DNA antisense AS or the oligonucleotides conjugates 1 or 4. As negative controls, the naked antisense and the free, uncoupled imidazole were used at concentrations and incubation times identical to those used for the conjugates (Material and Methods section). Alkaline hydrolysis and RNase T1 and U2 digestions of the RNA allow the positioning of the cleavage sites. One-hour incubation at the lower concentration (0.5 μM) is required to observe a significant cleavage at the expected location (Figure 4, arrow) only for the 5′-coupled imidazole conjugate 1. Three strong additional cleavages are observed for conjugates 1 and 4 (stars in Figure 4). With the naked oligonucleotide AS, these cleavages are very weak but can also be detected.Figure 4.

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