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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

Aptamers binding to the 5′-UTR of HIV-1.Autoradiograms of representative native polyacrylamide gels resolving binding assays of LXIV22 and LXIV26 (A), RNApt16 and RNApt16neg (B), to the UTR308. : unbound aptamer. : aptamer-target complexes. Mean values of three independent experiments are represented on the right.
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f5: Aptamers binding to the 5′-UTR of HIV-1.Autoradiograms of representative native polyacrylamide gels resolving binding assays of LXIV22 and LXIV26 (A), RNApt16 and RNApt16neg (B), to the UTR308. : unbound aptamer. : aptamer-target complexes. Mean values of three independent experiments are represented on the right.

Mentions: Binding to the HIV-1 target UTR308 was analyzed for the two most abundant aptamers in populations XI and XIV (represented by sequences XIV22 and XIV26), and for RNApt16. Since further inhibition assays were performed using fused aptamer-U6 snRNA cassette molecules, we carried out binding assays with these chimeric molecules, termed LXIV22 and LXIV26. RNApt16 was assayed without any modification for both binding and inhibitory assays, as we were interested in studying the functional properties of the minimal aptamer. The interaction of the RNApt16neg molecule with the target was also assayed as a negative control. The binding efficiency was analyzed by gel electrophoresis mobility shift assays (Fig. 5). Multiple complex conformers were observed for the LXIV22 aptamer RNA, whereas a major complex conformer was obtained for LXIV26 (Fig. 5A) and RNApt16 aptamers (Fig. 5B). No interaction was detected for the control molecule RNApt16neg, thus demonstrating the usefulness of our in silico approach for designing such a negative control. Results obtained from three independent experiments were quantified and fitted to a hyperbolic one-site binding curve with R2 coefficient higher than 0.99 for aptamers LXIV26 and RNApt16, yielding a Kd of 82 ± 13 nM and 280 ± 60 nM, respectively. Binding of the aptamer LXIV22 responded to a one-site specific binding curve with Hill slope, also with R2 coefficient higher than 0.99 and with a Kd of 154 ± 5 nM.


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)

Aptamers binding to the 5′-UTR of HIV-1.Autoradiograms of representative native polyacrylamide gels resolving binding assays of LXIV22 and LXIV26 (A), RNApt16 and RNApt16neg (B), to the UTR308. : unbound aptamer. : aptamer-target complexes. Mean values of three independent experiments are represented on the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Aptamers binding to the 5′-UTR of HIV-1.Autoradiograms of representative native polyacrylamide gels resolving binding assays of LXIV22 and LXIV26 (A), RNApt16 and RNApt16neg (B), to the UTR308. : unbound aptamer. : aptamer-target complexes. Mean values of three independent experiments are represented on the right.
Mentions: Binding to the HIV-1 target UTR308 was analyzed for the two most abundant aptamers in populations XI and XIV (represented by sequences XIV22 and XIV26), and for RNApt16. Since further inhibition assays were performed using fused aptamer-U6 snRNA cassette molecules, we carried out binding assays with these chimeric molecules, termed LXIV22 and LXIV26. RNApt16 was assayed without any modification for both binding and inhibitory assays, as we were interested in studying the functional properties of the minimal aptamer. The interaction of the RNApt16neg molecule with the target was also assayed as a negative control. The binding efficiency was analyzed by gel electrophoresis mobility shift assays (Fig. 5). Multiple complex conformers were observed for the LXIV22 aptamer RNA, whereas a major complex conformer was obtained for LXIV26 (Fig. 5A) and RNApt16 aptamers (Fig. 5B). No interaction was detected for the control molecule RNApt16neg, thus demonstrating the usefulness of our in silico approach for designing such a negative control. Results obtained from three independent experiments were quantified and fitted to a hyperbolic one-site binding curve with R2 coefficient higher than 0.99 for aptamers LXIV26 and RNApt16, yielding a Kd of 82 ± 13 nM and 280 ± 60 nM, respectively. Binding of the aptamer LXIV22 responded to a one-site specific binding curve with Hill slope, also with R2 coefficient higher than 0.99 and with a Kd of 154 ± 5 nM.

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