LNA™ should be used in experiments where increased affinity and specificity over standard DNA probes is critical or increased robustness is required. Incorporation of LNA™ into oligonucleotide probes gives you the freedom to modulate the probe design in respect to melting properties and hybridisation specificity. However, certain design rules are recommended because of the high affinity introduced by LNA™:
1) LNA™ hybridises very tightly when several consecutive residues are substituted with LNA™. Stretches of more than 4 LNA™s should be avoided, except for very short probes (9-10 nucleotides).
2) LNA™ will bind very tightly to other LNA™ residues, so prevention of self-complimentarity and cross-hybridisation to other LNA™ containing oligonucleotides in the assay is critical.
For very specific or novel assay settings, design rules may have to be established empirically, but following the above recommendations and using Exiqon’s LNA™ design tools will give you the best opportunities for using LNA™ probes to their full potential.
