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Published online 3 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 2 819-827
© 2004 Oxford University Press

Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays

Marcel Ooms, Koen Verhoef1, Edwin Southern1, Hendrik Huthoff and Ben Berkhout*

Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands and 1 Department of Biochemistry, University of Oxford, Oxford, UK

*To whom correspondence should be addressed. Tel: +31 20 566 4822; Fax: +31 20 566 6064; Email: b.berkhout{at}amc.uva.nl

Scanning arrays of antisense DNA oligonucleotides provide a novel and systematic means to study structural features within an RNA molecule. We used this approach to probe the structure of the untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This 335 nt RNA encodes multiple important replication signals and adopts two mutually exclusive conformations. The poly(A) and the dimer initiation signal (DIS) sequences of the leader RNA are base-paired in the long-distance interaction (LDI) conformation, but both domains form distinct hairpins in the branched multiple hairpins (BMH) conformation. An RNA switch mechanism has been proposed to regulate the activity of the DIS dimerization signal that is masked in one, yet exposed in the other conformation. The two RNA conformations demonstrate discrete differences in the array-based hybridization patterns. LDI shows increased hybridization in the poly(A) region and decreased hybridization in the DIS region when compared with BMH. These results provide additional evidence for the structure models of the two alternative leader RNA conformations. We also found a correlation between the efficiency of oligonucleotide hybridization and the accessibility of the RNA structure as determined by chemical and enzymatic probing in previous studies. The array approach therefore provides a very sensitive method to detect structural differences in related transcripts.


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