Locked Nucleic Acid (LNA™) is an RNA derivative in which the ribose ring is constrained by methylene linkage between the 2'-oxygen and 4'-carbon (Figure 1). This conformational restriction increases binding affinity for complementary sequences and provides an exciting new chemical approach to optimize and fine tune primers and probes for sensitive and specific detection of nucleic acids.

 

LNA can be mixed with DNA, RNA as well as other nucleic acid analogs using standard phosphoramidite DNA synthesis chemistry. Therefore, LNA oligonucleotides can easily be tagged with e.g. amino-linkers, biotin, fluorophores, etc. Thus a very high degree of freedom in the design of primers and probes exists. In order to facilitate efficient design of LNA containing primers and probes software tools have been developed for Tm prediction which can be accessed at www.LNA-tm.com.

Figure 2 demonstrates how both specificity (ΔTm) and Tm may be enhanced and adjusted for compatibility by including LNA. In the depicted example two capture probes targeting each of the two alleles in a SNP (Single Nucleotide Polymorphism) were enhanced by LNA. Please notice the increased Tm and ΔTm.

Figure 2. Fine-tuning of specificity (ΔTm) and Tm. The sequences below the bars represent the probes. The underlined positions indicate the site of the SNP and the nucleotides in red represent LNA monomers.