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Published online 4 June 2004

Nucleic Acids Research, 2004, Vol. 32, No. 10 3101-3107
© 2004 Oxford University Press

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1

Fabien Darfeuille1,2, Jens Bo Hansen3, Henrik Orum3, Carmelo Di Primo*,1,2 and Jean-Jacques Toulmé1,2

1 INSERM U386, Université Victor Segalen, 33076 Bordeaux cédex, France, 2 Institut Européen de Chimie et Biologie, 2 rue Escarpit, 33607 Pessac cédex, France and 3 Santaris Pharma A/S, Bøge Allé 3, DK-2970 Copenhagen, Denmark

*To whom correspondence should be addressed at Institut Européen de Chimie et Biologie, 2 rue Escarpit, 33607 Pessac cédex, France. Tel: +33 5 40 00 30 50; Fax: +33 5 40 00 30 45; Email: c.diprimo{at}iecb.u-bordeaux.fr
Present address:
Fabien Darfeuille, Department of Cell and Molecular Biology, Microbiology Program, Biomedical Center, Uppsala University, Box 596, S-751 24 Uppsala, Sweden

Received February 27, 2004; Revised April 26, 2004;; Accepted May 13, 2004

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera–TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.


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