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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

Ex vivo HIV-1 inhibition assays.(A) Schematic representation of the U6-aptamer RNA constructs. (B) Inhibition of HIV-1 p24 antigen production by LXIV22 and LXIV26, using ‘L-empty' as control. (C) Inhibition by RNApt16 and RNApt16neg. Data represent the mean of three independent experiments. **: statistically significant differences as compared to the control (ANOVA, p < 0.01).
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f6: Ex vivo HIV-1 inhibition assays.(A) Schematic representation of the U6-aptamer RNA constructs. (B) Inhibition of HIV-1 p24 antigen production by LXIV22 and LXIV26, using ‘L-empty' as control. (C) Inhibition by RNApt16 and RNApt16neg. Data represent the mean of three independent experiments. **: statistically significant differences as compared to the control (ANOVA, p < 0.01).

Mentions: The effect of the two most represented in vitro selected aptamers, XIV22 and XIV26, on HIV-1 production was assayed. Unmodified, pre-synthesized aptamers gave no satisfactory results (data not shown), probably due to their limited half-life or incorrect cellular location. Thus, we decided to flank them with the 5′ and 3′ stable hairpin-loop domains of the human U6 snRNA33 (Fig. 6A), thus giving rise to the modified aptamers termed LXIV22 and LXIV26. Aptamer sequences were cloned into vector pU614 and templates for in vitro transcription of aptamers LXIV22 or LXIV26 were prepared from the resulting plasmids. HEK 293T cells were co-transfected with 500 ng of in vitro transcribed LXIV22 or LXIV26 RNA molecules and 100 ng of plasmid pNL4.3 (containing the HIV-1 NL4.3 proviral DNA). Viral particle production was measured as p24 antigen abundance in the cellular supernatant, yielding inhibitions of 77 ± 7% and 80 ± 3%, respectively, in the HIV-1 viral particle production with respect to the control RNA termed ‘L-empty', that was transcribed from a pU6 plasmid without any cloned aptamer sequence, thus consisting in the two flanking U6 terminal 5′- and 3′-hairpins (Fig. 6B).


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)

Ex vivo HIV-1 inhibition assays.(A) Schematic representation of the U6-aptamer RNA constructs. (B) Inhibition of HIV-1 p24 antigen production by LXIV22 and LXIV26, using ‘L-empty' as control. (C) Inhibition by RNApt16 and RNApt16neg. Data represent the mean of three independent experiments. **: statistically significant differences as compared to the control (ANOVA, p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Ex vivo HIV-1 inhibition assays.(A) Schematic representation of the U6-aptamer RNA constructs. (B) Inhibition of HIV-1 p24 antigen production by LXIV22 and LXIV26, using ‘L-empty' as control. (C) Inhibition by RNApt16 and RNApt16neg. Data represent the mean of three independent experiments. **: statistically significant differences as compared to the control (ANOVA, p < 0.01).
Mentions: The effect of the two most represented in vitro selected aptamers, XIV22 and XIV26, on HIV-1 production was assayed. Unmodified, pre-synthesized aptamers gave no satisfactory results (data not shown), probably due to their limited half-life or incorrect cellular location. Thus, we decided to flank them with the 5′ and 3′ stable hairpin-loop domains of the human U6 snRNA33 (Fig. 6A), thus giving rise to the modified aptamers termed LXIV22 and LXIV26. Aptamer sequences were cloned into vector pU614 and templates for in vitro transcription of aptamers LXIV22 or LXIV26 were prepared from the resulting plasmids. HEK 293T cells were co-transfected with 500 ng of in vitro transcribed LXIV22 or LXIV26 RNA molecules and 100 ng of plasmid pNL4.3 (containing the HIV-1 NL4.3 proviral DNA). Viral particle production was measured as p24 antigen abundance in the cellular supernatant, yielding inhibitions of 77 ± 7% and 80 ± 3%, respectively, in the HIV-1 viral particle production with respect to the control RNA termed ‘L-empty', that was transcribed from a pU6 plasmid without any cloned aptamer sequence, thus consisting in the two flanking U6 terminal 5′- and 3′-hairpins (Fig. 6B).

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