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Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies.

Sánchez-Luque FJ, Stich M, Manrubia S, Briones C, Berzal-Herranz A - Sci Rep (2014)

Bottom Line: The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop.Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'.The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biology. Instituto de Parasitología y Biomedicina "López-Neyra" (IPBLN-CSIC), PTS Granada. Avda. del Conocimiento s/n, Armilla (Granada 18016, Spain) [2].

ABSTRACT
The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

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Related in: MedlinePlus

Model of putative functional effects of RNApt16 binding to poly(A) domain.UTR308 region in genomic (A) and subgenomic (B) RNAs is shown in black. The RNApt16 is shown in red. Poly(A)-gag pseudoknot is represented by a gray line in (A). The arrowhead points the major splicing donor.
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f7: Model of putative functional effects of RNApt16 binding to poly(A) domain.UTR308 region in genomic (A) and subgenomic (B) RNAs is shown in black. The RNApt16 is shown in red. Poly(A)-gag pseudoknot is represented by a gray line in (A). The arrowhead points the major splicing donor.

Mentions: The preferred target site of the selected aptamers and the RNApt16 molecule was the 5′-CUUGCC-3′ sequence (nts 81–86 of the HIV-1 genome) exposed in the apical loop of the essential poly(A) domain (Fig. 1). This hexamer is highly conserved among all the HIV-1 strains, subtypes, circulating intersubtype recombinant forms and groups, and even among the closely related simian immunodeficiency virus (SIV) from chimpanzee4041 (Fig. S9 and data not shown). This sequence conservation makes interesting to analyze the inhibitory potential of the selected aptamers and RNApt16 against different clinical HIV-1 isolates belonging to distinct viral subtypes and groups. Although an identical poly(A) domain is present at both ends of all intracellular genomic and subgenomic HIV-1 RNAs, only the poly(A) at the 3′ end leads to polyadenylation of the HIV RNAs42. Therefore, we hypothesize that the achieved inhibitory effect of the in vitro selected aptamers and the in silico derived one (RNApt16) might be explained as a result of the interference with the proper 3′ end RNA polyadenylation (Fig. 7).


Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies.

Sánchez-Luque FJ, Stich M, Manrubia S, Briones C, Berzal-Herranz A - Sci Rep (2014)

Model of putative functional effects of RNApt16 binding to poly(A) domain.UTR308 region in genomic (A) and subgenomic (B) RNAs is shown in black. The RNApt16 is shown in red. Poly(A)-gag pseudoknot is represented by a gray line in (A). The arrowhead points the major splicing donor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150108&req=5

f7: Model of putative functional effects of RNApt16 binding to poly(A) domain.UTR308 region in genomic (A) and subgenomic (B) RNAs is shown in black. The RNApt16 is shown in red. Poly(A)-gag pseudoknot is represented by a gray line in (A). The arrowhead points the major splicing donor.
Mentions: The preferred target site of the selected aptamers and the RNApt16 molecule was the 5′-CUUGCC-3′ sequence (nts 81–86 of the HIV-1 genome) exposed in the apical loop of the essential poly(A) domain (Fig. 1). This hexamer is highly conserved among all the HIV-1 strains, subtypes, circulating intersubtype recombinant forms and groups, and even among the closely related simian immunodeficiency virus (SIV) from chimpanzee4041 (Fig. S9 and data not shown). This sequence conservation makes interesting to analyze the inhibitory potential of the selected aptamers and RNApt16 against different clinical HIV-1 isolates belonging to distinct viral subtypes and groups. Although an identical poly(A) domain is present at both ends of all intracellular genomic and subgenomic HIV-1 RNAs, only the poly(A) at the 3′ end leads to polyadenylation of the HIV RNAs42. Therefore, we hypothesize that the achieved inhibitory effect of the in vitro selected aptamers and the in silico derived one (RNApt16) might be explained as a result of the interference with the proper 3′ end RNA polyadenylation (Fig. 7).

Bottom Line: The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop.Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'.The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biology. Instituto de Parasitología y Biomedicina "López-Neyra" (IPBLN-CSIC), PTS Granada. Avda. del Conocimiento s/n, Armilla (Granada 18016, Spain) [2].

ABSTRACT
The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

Show MeSH
Related in: MedlinePlus