1. Niels Tolstrup et al. summarized by Ki-Roo Shin
OligoDesign: optimal design of LNA (locked nucleic acid) oligo-nucleotide capture probes for gene expression profiling
Niels Tolstrup et al.
summarized by Ki-Roo Shin

2. Locked Nucleic Acids
a novel class of nucleic acid analogues. LNA monomers are bicyclic compounds structurally similar to RNA nucleosides.
The term "Locked Nucleic Acid" has been coined to emphasize that the furanose ring conformation is restricted in LNA by a methylene linker that connects the 2'-O position to the 4'-C position (see figure). By convenience, all nucleic acids containing one or more LNA modifications are called LNA.
LNA oligomers obey Watson-Crick base pairing rules and hybridize to complementary oligo-nucleotides. LNA provides vastly improved hybridization performance when compared to DNA and other nucleic acid derivatives in a number of situations.

3. Structure

4. Thermal Stability of LNA
LNA/DNA or LNA/RNA duplexes have increased thermal stability compared with similar duplexes formed by DNA or RNA. LNA has the highest affinity towards complementary DNA and RNA ever reported. In general, the thermal stability of a LNA/DNA duplex is increased 3°C to 8°C per modified base in the oligonucleotide.
The LNA modification has been shown to increase the biological stability of nucleic acids. Fully modified LNA oligonucleotides are resistant towards most nucleases tested.

5. Application Capture probes Sample preparation (mRNA) SNP analysis
Mutation analysis
Allele specific PCR
Antisense Target Validation
Hybridization probes
Strand invasion
In-situ hybridization
Triple-helix forming oligos
Nuclease protection assays

6. Advantages Affinity Tm modulation Specificity
LNA:LNA duplex formation constitutes the most stable Watson-Crick base pairing system yet developed.
LNA:LNA > LNA:RNA > RNA:RNA > RNA:DNA > DNA:DNA
Tm modulation
It is possible to fine-tune the placement of LNA bases to reach the desired Tm level without losing specificity.
Specificity
LNA enhances hybridization performance relative to native DNA and RNA, phosphorothiate or peptide nucleic acid (PNA) probes.
LNA lowers experimental error rates due to better mismatch discrimination.
LNA provides more robust assay conditions.
LNA improves signal-to-noise ratio.
Simplicity & Design Flexibility

7. OligoDesign Program BLAST analysis
Prediction of duplex thermal stability and Tm equalization using LAN substitutions
Self-annealing
Prediction of the secondary structures

8. Experimental Results C. elegans microarray
120 potential marker genes for a veriety of stress and toxicity processes and toxicologically relevant pathways, including drug metabolism, DNA damage-repair, apoptosis, stress response, membrane proteins and cell cycle regulators.
Design 50mer LNA substituted oligonuleotides
Two oligonucleotides showed high cross-hybridization signals to each other due to the high similarity (80%)

9. References
Koshkin,A. et al. (1998) LNA: synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron, 54,
Singh,S.K. et al. (1998) LNA: Synthesis and high-affinity nucleic acid recognition. Chem. Commun., 4,
T.Koch (2003) LNA: a family of high affinity nucleic acid probes. J. Phys.: Condensed Matter, 15, S1861-S1871