Application of RNA aptamers to the control of Application of RNA - PowerPoint PPT Presentation

Application of RNA aptamers to the control of Application of RNA aptamers to the control of the hepatitis C virus-CRE region function Alba Fernández-Sanlés † , Beatriz Berzal-Herranz † , Rodrigo González-Matamala, Pablo Ríos-Marco, Alfredo Berzal-Herranz * and Cristina Romero-López * † Equal contribution Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC. Av. Conocimiento s/n. PTS Granada, 18016. Armilla (Granada), Spain * Corresponding author: aberzalh@ipb.csic.es; cristina_romero@ipb.csic.es 1

A PPLICATION OF RNA A PTAMERS TO THE C ONTROL OF THE H EPATITIS C V IRUS -CRE R EGION F UNCTION HCV RNA genome CRE CRE IIIb II 5BSL3.2 IIIa Replication >90% IIIc IIId 5BSL3.1 5BSL3.3 3’X-tail PK2 3’SLIII 5’ Alt PolyU/UC PK1 3’ ORF I IV 3’SLII 3’SLII Stop codon AUG start codon HV 3’SLI IRES IRES 3’UTR 2

Abstract: Hepatitis C virus is an enveloped, ssRNA virus, which infects 3% of the world population. No fully efficient therapy for treating hepatitis C exists. This is mainly due to the quasispecies structure of the RNA genome population, which favors the emergence of resistant viral variants. Despite the high variability rate, significant sequence and, more importantly, structure conservation can be g y g q p y found in the so-called functional genomic RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are potential therapeutic targets. This study validates the use of RNA-based inhibitors (aptamers) as molecular tools to control the activity of the cis -acting replication element (CRE) within the HCV genome. The CRE is an essential partner g ( ) g for viral replication. Also this structural domain is involved in the regulation of the protein synthesis. A set of forty-four RNA aptamers was assayed for the ability to interfere with the viral RNA synthesis in a subgenomic replicon system. Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with specific and well-defined functional RNA domains of the CRE, yielding a decrease in the HCV genomic RNA levels higher than 90%. Concomitantly, one of them also promoted a significant increase in viral translation (>50%), likely by its interaction with the nucleotides surrounding the viral stop translation codon. The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE, thus explaining their antiviral activity. Present findings confirm the potential of the CRE as an anti-HCV drugs target and support the use of aptamers as molecular tools for challenging the functionality of RNA domains in viral genomes. Keywords: RNA aptamers; antiHCV Aptamers; HCV genome; CRE; 5BSL3.2; functional RNA domains. 3

RNA A PTAMER S ELECTION Target RNA-biotin Cloning, sequencing and analysis Sy Synthetic oligonucleotides Hi-Trap streptavidine Annealing Transcription chromatography and extension column Target Target recycling Aptamers are oligonucleotides able to recruit a wide variety of ligands. Aptamers are isolated from a SELEX (Systematic Evolution of Ligands by In vitro selection In vitro selection Exponential enrichment) process, which consists on iterative cycles of synthesis, binding, positive selection and amplification steps over a randomized oligonucleotide pool. The resulting population is enriched in those molecules able to bind to the desired target molecule. The highly dynamic folding of nucleic acids is the key to understand the specific and efficient interaction of aptamers to their cognate target, thus demonstrating the versatility and flexibility of nucleic acids of nucleic acids. Introduction 4

F UNCTIONAL RNA D OMAINS WITHIN THE HCV G ENOME AND L ONG -D ISTANT RNA-RNA C ONTACTS The isolation of aptamers directed against different protein targets of the hepatitis C virus (HCV) has been largely The isolation of aptamers directed against different protein targets of the hepatitis C virus (HCV) has been largely described. The HCV genome is a (+)ssRNA molecule encoding a single open reading frame (ORF) flanked by untranslated regions (UTRs), which are essential for viral replication, translation and infectivity. Conserved functional RNA domains have also been identified within the coding region, such as the cis -acting replication element (CRE), which is defined by three stem-loops, 5BSL3.1, 5BSL3.2 and 5BSL3.3. The 5BSL3.2 domain is critical for efficient HCV replication and the regulation of iral protein s nthesis These f nctional feat res depend on the establishment of long distant RNA RNA interactions of viral protein synthesis. These functional features depend on the establishment of long-distant RNA-RNA interactions with ith other genomic RNA domains. In addition, the 5BSL3.2 domain interacts with viral and host protein factors. CRE IIIb VI II NS5B eIF3 IIIa IIIc 5BSL3.2 40S EWSR1 IIId 5BSL3 3 5BSL3.3 5BSL3 1 5BSL3.1 3’X-tail PK2 3’SLIII 5’ Alt PolyU/UC PK1 3’ ORF I I IV 5BSL3.4 3’SLII 3’SLI Stop codon AUG start codon V HV HV IRES 3’UTR NS5B, viral RNA-dependent RNA polymerase; Introduction ESW3, Ewing sarcoma RNA-binding protein 1 5

A NTI -HCV RNA A PTAMER S ELECTION AGAINST THE CRE R EGION T7p PBS R PBS DNA cDNA RT-PCR In vitro o transcription RNA RNA RNA Elución RNA Active molecules Binding Selection PBS, primer binding site; Inactive R, randomized sequence; molecules T7p, T7 promoter Introduction 6

A NTI -HCV RNA A PTAMER S ELECTION AGAINST THE CRE R EGION APTAMER SEQUENCE (5 ′ - 3 ′ ) GROUP After six rounds of selection, a set containing forty-four different aptamers P6-1 CGUGGACGAGAGCUGGUA GUG UGUGGCGAU 1 4 isolated for their binding ability to the CRE was assayed for their capacity g y y p y P6-2 GCUGCUGUUACGUACUAAGGUGCGGCGGGG 5 to interfere with the CRE functionality: replication, translation and binding P6-6 CGGCUCUGGAUGGCGCUGUUUGUGUGUGGU 4 P6-8 CAUUGUGCGACUGGGAGAGGGCGUGUCCG 3 to the viral RNA-dependent RNA polymerase (NS5B). All of them beared P6-19 CGUCCCGGCUGCGACAGGAUGGGGACAUGG 2 sequence motifs (the so-called consensus motifs, indicated as groups 1-5) P6-20 CAACGUGGCGAUGGCGUGUGUACGAUGUGG 4 complementary to different conserved elements whithin the HCV CRE. P6-23 CGUGUGCGCAG UGG GCAUCUGCGGACAGGG 1 3 4 P6-43 GCAUCGGUGGGUAUUGCAGUGCCCGGCUGU 2 3 P6 44 P6-44 CGCGGCUUUGGGGACGUUAGCCAUCUGAUG CGCGGCUUUGGGGACGUUAGCCAUCUGAUG 5 5 P6-45 CGUGUGUGCUGGCUAGUGGUGAGUCCGG 1 4 5BSL3.1 P6-50 CGGAGGUU G UG UG GGGGACGUCUGUUGUGC 2 4 5 U U U A P6-53 CAGGUGGUGUUAGUUACGCGUAGGCGUGCC 1 A U 9240 G C P6-57 GCGGCCUGCGAUCUGGAUGCUGCGUGGGCC 3 5BSL3.3 G C U A P6-64 CCGAGGU GG CUGGGGACAGCAGGAGGAGCG 1 2 U U U G C G C U U 5BSL3.4 5BSL3.4 P6-76 GGCAGCUCUAGAGGGGGCGUAAUCGGCUCG 3 A A U U C G C G P6-77 GUGCUUGCGGUGUUGAGCCCAGCGGUAGUG 1 A U A 9340 U G C G U A G C C U P6-78 GGUACGGCAUGGCGCUACGGCUGGAUCGUG 2 G C A U U C C G G C A P6-79 GCUAUGGUGGCCUGGUCCGUCGGGGGGCCG 1 5 U G A C G C A C G G U P6-80 CGCUAGUGUGGCGUGUUGCAGUAGGCAGAG 4 C G A U U U C G A C G U 9220 C G P6-81 CAGGAUGAGUACUGGGCUCCUCGGCGUUGG 3 G C C G Group 4 G U C G C G 9380 C G P6-82 GUGUGUAUGCAUUGACGGACGACUGGCCGG 4 G C U A 5’ 5’ G G G G G G C C A A G G U U A A A A G G G G A A C C C C A A A A G G C C U U C C A A A A A A C C U U C C C C A A U U C C C C A A A A U U C C C C G G U U U U A A 3’ 3’ G G UU UU C C A A U U G G U U G G G G U U A A UC A UC A U U U U CUAC CUAC G G C C C G I I I P6-83 GGUGGAUUGGUGACCUUUGUGCUACGGGCA 1 3 I I A U 9181 9200 9260 9320 Group 1 9360 G C Group 5 P6-84 CCCUGUGUUGGGCGGGCUACGUGUGUGGAG 3 4 C G G C P6-85 GGGGCGUGUUCGGGACGCCUUGUACGAACG 3 G C P6-86 CGGGCGUCGACUGAAGUUUGAGGUGAAGGA 1 3 G C 9300 G C I A G C P6-87 CGUGAUAGUUGUGCUGGCCGAUG GGU GGAC 1 3 Group 3 C C P6-88 CGCUGG UGG GUAGAGGUGUUUGUGUGCUGU 1 3 4 G UG G C P6 89 P6-89 CGCCGUGCCAGCUCGGGACGGUGCGGCAGG CGCCGUGCCAGCUCGGGACGGUGCGGCAGG 3 3 A U A U G C P6-91 GUCGGCUGUUGACACGUGUAGUGUG GGU GG 1 3 4 A U C G P6-94 CGCAGUGGAGGGCGAAUAAGAAUGUGACAG 1 3 A U U C P6-95 CGGUGUGCGUGUGGGGACGCGUUCGUACAG 4 5 A C Group 2 U G P6-96 CGUGUUACGGCUGUGCUGGGUACAUCGGUG 2 3 A A 9280 U AC P6-98 GAUGAGGCCUCGGUAGUGUGGACAGUGCAG 4 C 5BSL3.2 P6-99 GUGGCCGUG UG GGCAACGGAACAUGCCGUG 3 4 P6-100 CGUGUUACGGCUGUGCUGGGUACAUCGGUG 2 P6-101 CGGUAACGUGGCCUUAGGGCAGGAGGCUG 2 3 P6-102 CGGCACGAUGUGUCUACCGCGG UGG GGC 1 4 5 P6-103 GGUUGGACGUCGUCUGUGGGGGACUCGUGC 2 5 Results and discussion 7