Design and characterization of decoy oligonucleotides containing locked nucleic acids.

Transfection of cis-element double-stranded oligonucleotides, referred to as decoy ODNs, has been reported to be a powerful tool that provides a new class of antigene strategies for gene therapy. However, one of the major limitations of the decoy approach is the rapid degradation of phosphodiester oligonucleotides by intracellular nucleases. To date, several DNA analogs have been employed to overcome this issue, but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this paper we have investigated the use of conformationally restricted nucleotides in the design of decoy molecules for nuclear transcription factor kappaB (NF-kappaB). Starting from a synthetic double-stranded oligonucleotide, containing the kappaB consensus binding sequence, we designed a panel of decoy molecules modified to various extents and at various positions with locked nucleic acids (LNAs). Our results indicate that the addition of terminal LNA bases, outside the kappaB sequence, to generate LNA-DNA-LNA co-polymers was sufficient to confer appreciable protection towards nuclease digestion, without interfering with transcription factor binding. Conversely, insertion of LNA substitutions in the context of the kappaB-binding site resulted in further increased stability, but caused a loss of affinity of NF-kappaB for the target sequence. However, our results also indicate that this latter effect was apparently dependent not only on the extent but also on strand positioning of the internal LNA substitutions. This observation is of great importance since it provides evidence for the possibility of tuning DNA-LNA duplexes with internal LNAs into decoy agents with improved features in terms of biological stability and inhibitory effect.

Transcription factor decoy oligonucleotides modified with locked nucleic acids: an in vitro study to reconcile biostability with binding affinity.

Double-stranded oligonucleotides (ODNs) containing the consensus binding sequence of a transcription factor provide a rationally designed tool to manipulate gene expression at the transcriptional level by the decoy approach. However, modifications introduced into oligonucleotides to increase stability quite often do not guarantee that transcription factor affinity and/or specificity of recognition are retained. We have previously evaluated the use of locked nucleic acids (LNA) in the design of decoy molecules for the transcription factor kappaB. Oligo nucleotides containing LNA substitutions displayed high resistance to exo- and endonucleolytic degradation, with LNA-DNA mix-mers being more stable than LNA-DNA-LNA gap-mers. However, insertion of internal LNA bases resulted in a loss of affinity for the transcription factor. This latter effect apparently depended on positioning of the internal LNA substitutions. Indeed, here we demonstrate that intra- and inter-strand positioning of internal LNAs has to be carefully considered to maintain affinity and achieve high stability, respectively. Unfortunately, our data also indicate that LNA positioning is not the only parameter affecting transcription factor binding, the interference in part being dependent on the intrinsic conformational properties of this nucleotide analog. To circumvent this problem, the successful use of an alpha-L-ribo- configured LNA is demonstrated, indicating LNA-DNA-alpha-L-LNA molecules as promising new decoy agents.