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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.This work was supported by the Spanish Ministerio de Ciencia e Innovación [BFU2009-08137 to A.B.-H., BIO2010-20696 to C.B. and FIS2011-27569 to S.M.]; by the Spanish Ministerio de Economía y Competitividad [BFU2012-31213 to A.B.-H.]; by the Junta de Andalucía [CVI-7430 to A.B.-H.]; by the Spanish National Research Council [201120E004 to A.B.-H.]; by Comunidad de Madrid [MODELICO, S2009/ESP-1691 to S.M.] and by FEDER funds from the EU to C.B. and A.B.-H. CIBERehd is funded by the Instituto de Salud Carlos III.Peer reviewe

Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies