Trends in Biotechnology
LNA: a versatile tool for therapeutics and genomics
Section snippets
Recognition of single-stranded DNA and RNA
The hybridization properties of LNA oligonucleotides have been evaluated in several different sequences, ranging from six to ∼20-nucleotide long oligomers with various levels of LNA content (for reports with comprehensive tables, see for example 5, 9, 14, 15). Hybridization of LNA with either DNA or RNA targets has demonstrated unprecedented thermostabilities, shown by remarkable increases in melting points per LNA monomer introduced (ΔTm values): ΔTm=+1 – +8°C against DNA 5, 6, 9, 14, 16 and ΔT
Molecular structures of LNA hybrids
Several LNA–RNA and LNA–DNA hybrids have been structurally characterized by NMR spectroscopy and X-ray crystallography. In general, these hybrids retain the features common for native nucleic acid duplexes (i.e. usual Watson–Crick base pairing, nucleobases in the anti orientation, base stacking and a right-handed helical conformation).
NMR spectroscopic studies of three nonamer LNA–RNA hybrids, in which the LNA strand contained one, three and nine LNA monomers, respectively 17, 29, revealed
LNA and therapeutic applications
The use of oligonucleotide analogues for regulation of gene expression is a concept eagerly pursued because of its enormous therapeutic potential 34, 35. In principle, an antisense oligonucleotide (AO), complementary to a given mRNA, is introduced into the cell and binds to the mRNA, thereby inhibiting translation. There are several mechanisms by which an AO might silence the message of the target mRNA (e.g. inhibition or alteration of splicing, translational arrest, redirection of
LNA in diagnostics
Single-nucleotide mutations in the genetic code are the cause of several ailments. The traditional methods of scanning for single-nucleotide polymorphisms (SNPs) are generally time-consuming and difficult to automate because enzymatic digestion and/or gel electrophoresis are required. Easy-to-use SNP assays based on the LNA technology have been designed and implemented. These assays rely on the observation that single-nucleotide mismatch discrimination is better for LNA than for DNA (i.e. the
LNA as aptamers (decoys)
Aberrant activation and expression of genes, whose products are involved in initiation and progression of pathogenesis, cause several diseases. In recent years, strategies targeting transcription-activating proteins have emerged. One such strategy is the use of dsDNAs bearing the consensus binding sequence of a specific transcription factor. Once transfected into cells, these dsDNA aptamers, dubbed decoys, interact with the target factor. Interaction with the decoy results in incapacitation of
Conclusions and outlook
LNA is a general and versatile tool for high-affinity recognition of ssDNA, ssRNA and dsDNA. Not only can fully modified LNA be applied but so can, for example, LNA/DNA, LNA/RNA, LNA/2′-OMe-RNA and LNA/phosphorothioate-DNA chimeras. This unique freedom of LNA design offers the possibility of fine-tuning the hybridization of virtually any oligonucleotide and also the use of known oligonucleotide modifications and technologies in combination with LNA.
Several reports have recently appeared on the
Acknowledgements
We are grateful to the many collaborators who have contributed to the development and study of LNA. We acknowledge financial support from the Danish National Research Foundation, The Danish Research Agency and Exiqon A/S.
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