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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

Schematic representation of the BMH conformation of the HIV-1 UTR308.Essential structural domains are indicated. Putative binding sites for RNApt16 (boxed) and the in vitro selected aptamers are shown in boldface, and labelled from (a) to (f). The alternative, partially overlapping binding region (a′) is shown.
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f1: Schematic representation of the BMH conformation of the HIV-1 UTR308.Essential structural domains are indicated. Putative binding sites for RNApt16 (boxed) and the in vitro selected aptamers are shown in boldface, and labelled from (a) to (f). The alternative, partially overlapping binding region (a′) is shown.

Mentions: The human immunodeficiency virus type 1 (HIV-1) genome is a 9.2 kb long, single-stranded RNA (ssRNA) molecule of positive polarity with multiple open reading frames (ORFs), including those for structural and functional proteins gag, pol and env. The coding region is flanked at both ends by untranslated regions (UTR). HIV-1 infective particles contain two identical genomic RNA molecules. The genome is reverse transcribed into a cDNA copy that is integrated into the host genome. The transcription of the proviral cDNA generates capped, full-length genomic RNAs that must undergo alternative splicing to accommodate the different ORFs immediately downstream of the capped 5′-end. All the HIV-1 genomic and subgenomic RNAs share approximately the first 300 nt of their 5′-UTR. This sequence contains several well-characterized and conserved functional RNA domains12 including the trans-activator response (TAR) element3, the polyadenylation [poly(A)] region4, the primer-binding site (PBS)5, the dimerisation initiation site (DIS)6, the major splicing donor (SD)7 and part of the packaging signal (psi)8 (Fig. 1). These RNA domains are involved in alternative, functionally relevant RNA-RNA interactions that determine two mutually exclusive conformations of the overall 5′-UTR region, termed Branched Multiple Hairpins (BMH) and Long Distance Interaction (LDI)9.


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)

Schematic representation of the BMH conformation of the HIV-1 UTR308.Essential structural domains are indicated. Putative binding sites for RNApt16 (boxed) and the in vitro selected aptamers are shown in boldface, and labelled from (a) to (f). The alternative, partially overlapping binding region (a′) is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Schematic representation of the BMH conformation of the HIV-1 UTR308.Essential structural domains are indicated. Putative binding sites for RNApt16 (boxed) and the in vitro selected aptamers are shown in boldface, and labelled from (a) to (f). The alternative, partially overlapping binding region (a′) is shown.
Mentions: The human immunodeficiency virus type 1 (HIV-1) genome is a 9.2 kb long, single-stranded RNA (ssRNA) molecule of positive polarity with multiple open reading frames (ORFs), including those for structural and functional proteins gag, pol and env. The coding region is flanked at both ends by untranslated regions (UTR). HIV-1 infective particles contain two identical genomic RNA molecules. The genome is reverse transcribed into a cDNA copy that is integrated into the host genome. The transcription of the proviral cDNA generates capped, full-length genomic RNAs that must undergo alternative splicing to accommodate the different ORFs immediately downstream of the capped 5′-end. All the HIV-1 genomic and subgenomic RNAs share approximately the first 300 nt of their 5′-UTR. This sequence contains several well-characterized and conserved functional RNA domains12 including the trans-activator response (TAR) element3, the polyadenylation [poly(A)] region4, the primer-binding site (PBS)5, the dimerisation initiation site (DIS)6, the major splicing donor (SD)7 and part of the packaging signal (psi)8 (Fig. 1). These RNA domains are involved in alternative, functionally relevant RNA-RNA interactions that determine two mutually exclusive conformations of the overall 5′-UTR region, termed Branched Multiple Hairpins (BMH) and Long Distance Interaction (LDI)9.

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