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Development of Optimized Inhibitor RNAs Allowing Multisite-Targeting of the HCV Genome

by Cristina Romero-López, Thomas Lahlali, Beatriz Berzal-Herranz and Alfredo Berzal-Herranz

Molecules 2017, 22(5), 861; https://doi.org/10.3390/molecules22050861 - 22 May 2017

Cited by 9 | Viewed by 4735

Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in [...] Read more.

Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome’s internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC₅₀ values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugates)

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Figure 1

Figure 1
The HCV IRES region and the parental HH363-10 chimeric inhibitor employed as the prototype for the construction of the RNA pool. (A) Sequence and secondary structure of the HCV IRES including the functional RNA domains targeted by the selected chimeric inhibitory RNA molecules. The IRES site cleaved by HH363-10 is indicated by an arrow. The translation start codon at position 342 is enlarged. The nucleotides that interact with the aptamer domain of HH363-10 are shown in red. Residues proposed to interact with the aptamer domain of HH-11 and HH-17 are shown in blue. The theoretical anchoring site for HH-26 is indicated in blue and underlined. Nucleotides pictured in green, located in the linker region between domains I and II, likely act as a binding region for the aptamer domain of the chimeric inhibitors HH-13, HH-22, HH-24, HH-28 and HH-43; (B) Sequence and theoretical secondary structure model of HH363-10. Figure was adapted from [<a href="#B14-molecules-22-00861" class="html-bibr">14</a>]. The catalytic domain, HH363, is shadowed. Tertiary contacts are indicated by dotted lines. Residues in the aptamer domain responsible for the interaction with domain IIIf of the IRES are shown in red. Randomization of the residues flanking this sequence motif, plus partial mutagenesis of those nucleotides participating in the IIIf binding site, yielded an initial population of more than 6 × 107 theoretical variants (lower panel). R, G or A; K, G or U; PK, pseudoknot. Full article ">Figure 2
Selected RNA molecules targeting the HCV IRES region. Sequences of the 10 selected chimeric inhibitory RNAs after seven rounds of selection. Residues in grey denote the conserved catalytic domain of the inhibitory RNA. The sequence motif involved in the interaction with the domain IIIf is indicated in red. The nucleotides theoretically targeting domain IV and the linker sequence between domains I and II within the IRES are pictured in blue and green respectively. The unique sequence in HH-26 that binds to the apical loop of IRES domain IV is also underlined. Full article ">Figure 3
Specific inhibition of HCV IRES-dependent translation in vitro by the selected chimeric inhibitory RNAs. (A) The bar chart shows the relative synthesis of FLuc protein directed by the HCV IRES region achieved in the presence of 5 µM of each chimeric inhibitory RNA, normalized to that obtained for the cap-dependent translation of cap-RLuc mRNA. The resulting values were referred to the results obtained in the absence of inhibitory RNA. Data are the mean of at least three independent assays ± standard deviation represented by the error bars; (B) The plot shows the relative reduction in FLuc synthesis caused by the chimeric inhibitors HH-11, HH-13, HH-26, HH-28 and HH-43. Data were normalized to those obtained for the synthesis of the RLuc reporter protein. The relative amount of FLuc obtained at each inhibitor concentration is calculated as a percentage with respect to the control reaction in the absence of any chimeric inhibitory RNA. Data (the mean of three independent assays ± standard deviation) were fitted to a non-linear regression curve to determine the IC50 values; (C) The histogram shows the effect of each anti-HCV inhibitory RNA on the IRES function of the closely related GBV-B virus. The synthesis of the FLuc protein was normalized as noted in A). Data are the mean of three independent assays ± standard deviation. Full article ">Figure 4
The selected chimeric inhibitory RNAs interfere with HCV translation and replication in a human hepatoma cell line. (A) Huh-7 cells were transfected with 5 µg of each chimeric inhibitory RNA or the non-related RNA80, plus 1.1 µg of a mixture containing the IRES-FLuc mRNA and the cap-RLuc. Luciferase activity was measured 18 h after transfection and the values turned into percentages by reference to the results obtained in the control assay with RNA80. Data are the mean of four independent experiments ± standard deviation represented by the error bars; (B) Five µg of the different chimeric inhibitory RNAs were used to transfect Huh-7 cells stably supporting HCV replication. Total RNA was extracted 24 h post-transfection using TRIzol™, and the relative amount of HCV RNA quantified by RT-qPCR. Data are the mean of four independent assays ± standard deviation represented by the error bars. Full article ">Figure 5
Theoretical model for the folding of the chimeric inhibitory RNAs HH-11, HH-13, HH-26, HH-28 and HH-43. In silico studies using the RNAstructure software [<a href="#B26-molecules-22-00861" class="html-bibr">26</a>] were performed to model the secondary structure of the RNA molecules under study; the figure shows the results obtained. The colour code is the same as that indicated in <a href="#molecules-22-00861-f002" class="html-fig">Figure 2</a>. Full article ">

3882 KiB

Open AccessArticle

CTAB-Assisted Fabrication of Bi₂WO₆ Thin Nanoplates with High Adsorption and Enhanced Visible Light-Driven Photocatalytic Performance

by Yuxue Zhou, Pengfei Lv, Xiangdong Meng, Yanping Tang, Pingping Huang, Xiaobing Chen, Xiaoshuang Shen and Xianghua Zeng

Molecules 2017, 22(5), 859; https://doi.org/10.3390/molecules22050859 - 22 May 2017

Cited by 19 | Viewed by 5665

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