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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

Aptamer sequences of rounds XI and XIV, grouped attending to putative common targets.The variable region is shown in boldface. The single-stranded sequences predicted by RNAfold software59 are boxed. White text represents the sequences complementary to 5′-UTR loop regions (shown below each group). x N: sequence multiplicity in each round. Among these putative interaction sites, once the energy of the folded configuration is considered (Table S2) the only robust and energetically favoured interaction is that with the poly(A) apical loop.
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f3: Aptamer sequences of rounds XI and XIV, grouped attending to putative common targets.The variable region is shown in boldface. The single-stranded sequences predicted by RNAfold software59 are boxed. White text represents the sequences complementary to 5′-UTR loop regions (shown below each group). x N: sequence multiplicity in each round. Among these putative interaction sites, once the energy of the folded configuration is considered (Table S2) the only robust and energetically favoured interaction is that with the poly(A) apical loop.

Mentions: An in vitro selection strategy was used to isolate anti-HIV-1 RNA aptamers targeting the first 308 nt of the 5′-UTR of the HIV-1 subtype B NL4.3 genome. This target corresponds to the sequence fragment of the 5′UTR that is common to all genomic and subgenomic HIV-1 RNAs2, including 8 nt from the unspliced SD domain that have been maintained in the molecule to prevent alternative foldings of the entire molecule due to SD partial deletion after splicing. The starting RNA population consisted of roughly 7 × 1015 variants of 64 nt long molecules. RNA molecules were binding-challenged against the target molecule fixed to a sepharose-streptavidin column. Fourteen cycles of selection-amplification were performed, and the selective pressure was stepwise modified by increasing the binding temperature from round IV on, and by reducing the target:aptamer ratio from round XI on. The yield of complex formation increased along the selection process, as shown by the evolution of Bmax32 (Fig. 2). A total of 299 individual clonal sequences derived from the populations selected at rounds 0 (initial random population, 30 seqs), I (30), III (31), V (24), VIII (28), IX (32), X (35), XI (52) and XIV (37) were analyzed (Fig. 3 and data not shown). The presence of intra- and/or inter-round repeated sequences supposed a reduction in the diversity of the analyzed collection to 216 different molecules (Table S1). The repeated sequences gained representation from round IX on, thus showing that the selection process was effective. Finally, 188 out of the 216 different sequences showed the expected length of 64 nt, while the others contained deletions of one or more nucleotides in their variable region. The clustering of those 188 different, 64 nt-long sequences, is shown in Figure S1.


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)

Aptamer sequences of rounds XI and XIV, grouped attending to putative common targets.The variable region is shown in boldface. The single-stranded sequences predicted by RNAfold software59 are boxed. White text represents the sequences complementary to 5′-UTR loop regions (shown below each group). x N: sequence multiplicity in each round. Among these putative interaction sites, once the energy of the folded configuration is considered (Table S2) the only robust and energetically favoured interaction is that with the poly(A) apical loop.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Aptamer sequences of rounds XI and XIV, grouped attending to putative common targets.The variable region is shown in boldface. The single-stranded sequences predicted by RNAfold software59 are boxed. White text represents the sequences complementary to 5′-UTR loop regions (shown below each group). x N: sequence multiplicity in each round. Among these putative interaction sites, once the energy of the folded configuration is considered (Table S2) the only robust and energetically favoured interaction is that with the poly(A) apical loop.
Mentions: An in vitro selection strategy was used to isolate anti-HIV-1 RNA aptamers targeting the first 308 nt of the 5′-UTR of the HIV-1 subtype B NL4.3 genome. This target corresponds to the sequence fragment of the 5′UTR that is common to all genomic and subgenomic HIV-1 RNAs2, including 8 nt from the unspliced SD domain that have been maintained in the molecule to prevent alternative foldings of the entire molecule due to SD partial deletion after splicing. The starting RNA population consisted of roughly 7 × 1015 variants of 64 nt long molecules. RNA molecules were binding-challenged against the target molecule fixed to a sepharose-streptavidin column. Fourteen cycles of selection-amplification were performed, and the selective pressure was stepwise modified by increasing the binding temperature from round IV on, and by reducing the target:aptamer ratio from round XI on. The yield of complex formation increased along the selection process, as shown by the evolution of Bmax32 (Fig. 2). A total of 299 individual clonal sequences derived from the populations selected at rounds 0 (initial random population, 30 seqs), I (30), III (31), V (24), VIII (28), IX (32), X (35), XI (52) and XIV (37) were analyzed (Fig. 3 and data not shown). The presence of intra- and/or inter-round repeated sequences supposed a reduction in the diversity of the analyzed collection to 216 different molecules (Table S1). The repeated sequences gained representation from round IX on, thus showing that the selection process was effective. Finally, 188 out of the 216 different sequences showed the expected length of 64 nt, while the others contained deletions of one or more nucleotides in their variable region. The clustering of those 188 different, 64 nt-long sequences, is shown in Figure S1.

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