Implications of RNA structure on the annealing of a potent antisense RNA directed against the human immunodeficiency virus type 1.
Antisense RNA-mediated regulation in bacterial systems is related to the kinetics of RNA-RNA annealing in vitro. Here, we investigated the secondary structure of alphaY69, an effective HIV-directed antisense RNA in human cells. Purified RNA preparations contain a single conformer. The global structure was identified by a cleavage experiment under native conditions using a short complementary oligonucleotide and RNase H. Structural analyses indicate a three-domain structure of alphaY69 consisting of two stem-loop elements connected by a seven-nucleotide single-stranded hinge region. Kinetic data suggest that the formation of base pairs between a CGC triplet of alphaY69 and its target RNA is essential for fast annealing. The complementary sequence stretch of the target folds into a high-energy secondary structure. The relationship between modifications in structural elements of alphaY69 and the annealing kinetics suggested that rate-limiting steps of the annealing involve a single site of alphaY69 and do not involve its 5' or 3'-end. Further, the data indicate that both initial base-specific interactions and duplex formation are dependent on the CGC triplet of the central region of alphaY69. This mechanism represents a specific and efficient way of RNA-RNA annealing that is initiated by the interaction of unstructured RNA regions.
Eckardt
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S
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany.
Romby
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P
Sczakiel
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G
D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000494:pharmacology; D016372:RNA, Antisense / Q000737:chemistry* / Q000494:pharmacology*; D012367:RNA, Viral / Q000737:chemistry* / Q000187:drug effects; D016914:Ribonuclease H; D006163:Ribonuclease T1; D012984:Software; D013816:Thermodynamics; D014158:Transcription, Genetic
D019380:Anti-HIV Agents; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D016372:RNA, Antisense; D012367:RNA, Viral; D016914:Ribonuclease H; D006163:Ribonuclease T1