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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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In silico prediction of the MFE secondary structure of the aptamers.(A) Group 2. (B) Group 1. (C) Rest of structures. (D) RNApt16 and RNApt16neg. _N: number of repetitions of a particular sequence. The 16 nt-long stem-loop motif is boxed. Probabilities for every nucleotide to actually hold the structural position shown are represented by a colour code from deep blue (lowest) to red (highest).
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f4: In silico prediction of the MFE secondary structure of the aptamers.(A) Group 2. (B) Group 1. (C) Rest of structures. (D) RNApt16 and RNApt16neg. _N: number of repetitions of a particular sequence. The 16 nt-long stem-loop motif is boxed. Probabilities for every nucleotide to actually hold the structural position shown are represented by a colour code from deep blue (lowest) to red (highest).

Mentions: The in silico predicted MFE secondary structure model of the aptamers selected in round XIV is depicted in Figure 4A–C. Folding energies corresponding to each structure (in Kcal/mol) are ΔG[XIV22] = −10.80; ΔG[XIV1] = −11.80; ΔG[XIV32] = −10.80; ΔG[XIV12] = −10.90; ΔG[XIV26] = −15.70; ΔG[XIV5] = −11.10; ΔG[XIV48] = −9.80; ΔG[XIV37] = −7.32; ΔG[XIV25] = −11.50. The most abundant structures (belonging to the Groups 1 and 2 defined above) indicate that the consensus octamer 5′-GGCAAGGA-3′ is always placed in an apical loop flanked by complementary sequences that form a double stranded region of at least 4 bp in length. The RNAfold algorithm calculates the likelihood that any nucleotide in a sequence actually occupies the predicted structural position by analysing how often it appears in the structural ensemble of the analysed sequence. While some of the motifs present in the folded sequences have low reliability, we observed that the consensus octamer forming a loop plus its 4 nt flanking regions consistently shows a stem-loop configuration. Such an RNA motif is systematically found in most structures of the thermodynamic ensemble of sequences in round XIV (Figure 4A–B), and does not contribute significantly to their observed ED. The consensus sequence of that conserved motif, with structure 5′-((((........))))-3′, is 5′-NNDYGGCARGGARNNN-3′ (sequence alignment not shown). Based on this fact, a 16 nt-long stem-loop RNA molecule was in silico designed as a minimal aptamer potentially able to interact with the 5′-UTR of HIV-1. This aptamer was termed RNApt16, and included the consensus octamer in a loop closed by the 4 bp-long stem allowing the highest possible thermodynamic stability of the folded molecule, thus formed by four consecutive C-G base pairs: 5′-CCCCGGCAAGGAGGGG-3′. The secondary structure of RNApt16 is shown in Figure 4D, the folding energy associated to its MFE is −6, 50 kcal/mol, and the frequency of this MFE within the thermodynamic ensemble is 91,82%.


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)

In silico prediction of the MFE secondary structure of the aptamers.(A) Group 2. (B) Group 1. (C) Rest of structures. (D) RNApt16 and RNApt16neg. _N: number of repetitions of a particular sequence. The 16 nt-long stem-loop motif is boxed. Probabilities for every nucleotide to actually hold the structural position shown are represented by a colour code from deep blue (lowest) to red (highest).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: In silico prediction of the MFE secondary structure of the aptamers.(A) Group 2. (B) Group 1. (C) Rest of structures. (D) RNApt16 and RNApt16neg. _N: number of repetitions of a particular sequence. The 16 nt-long stem-loop motif is boxed. Probabilities for every nucleotide to actually hold the structural position shown are represented by a colour code from deep blue (lowest) to red (highest).
Mentions: The in silico predicted MFE secondary structure model of the aptamers selected in round XIV is depicted in Figure 4A–C. Folding energies corresponding to each structure (in Kcal/mol) are ΔG[XIV22] = −10.80; ΔG[XIV1] = −11.80; ΔG[XIV32] = −10.80; ΔG[XIV12] = −10.90; ΔG[XIV26] = −15.70; ΔG[XIV5] = −11.10; ΔG[XIV48] = −9.80; ΔG[XIV37] = −7.32; ΔG[XIV25] = −11.50. The most abundant structures (belonging to the Groups 1 and 2 defined above) indicate that the consensus octamer 5′-GGCAAGGA-3′ is always placed in an apical loop flanked by complementary sequences that form a double stranded region of at least 4 bp in length. The RNAfold algorithm calculates the likelihood that any nucleotide in a sequence actually occupies the predicted structural position by analysing how often it appears in the structural ensemble of the analysed sequence. While some of the motifs present in the folded sequences have low reliability, we observed that the consensus octamer forming a loop plus its 4 nt flanking regions consistently shows a stem-loop configuration. Such an RNA motif is systematically found in most structures of the thermodynamic ensemble of sequences in round XIV (Figure 4A–B), and does not contribute significantly to their observed ED. The consensus sequence of that conserved motif, with structure 5′-((((........))))-3′, is 5′-NNDYGGCARGGARNNN-3′ (sequence alignment not shown). Based on this fact, a 16 nt-long stem-loop RNA molecule was in silico designed as a minimal aptamer potentially able to interact with the 5′-UTR of HIV-1. This aptamer was termed RNApt16, and included the consensus octamer in a loop closed by the 4 bp-long stem allowing the highest possible thermodynamic stability of the folded molecule, thus formed by four consecutive C-G base pairs: 5′-CCCCGGCAAGGAGGGG-3′. The secondary structure of RNApt16 is shown in Figure 4D, the folding energy associated to its MFE is −6, 50 kcal/mol, and the frequency of this MFE within the thermodynamic ensemble is 91,82%.

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