Locked Nucleic Acids (LNAs)

LNAs are a novel chemical modification to the sugar backbone that can be incorporated into oligonucleotides. The LNA modification introduces a 2´-O, 4´-C methylene bridge in the sugar ring as shown below. This bridge serves to "lock" the sugar backbone into the 3´-endo conformation, which significantly increases Tm (i.e. stabilizes the duplex in A-form conformation) as well as provides resistance to nuclease degradation.

LNA bases can be incorporated into oligos as short all-LNA sequences or as longer LNA/DNA chimeras. LNAs can be placed in internal or 5´-positions. We do not offer 3´-LNA at the present time; an LNA base can be incorporated as the first 3´-base if a modified support is used, such as 3´-phos.

When inserting LNA bases into a nucleic acid sequence, include the prefix "+" immediately before the desired base substitution. For example, "+A" = LNA-A base.

The "codes" to insert various modified backbones with "ATCG" sequence into an oligo are as follows:

When ordering an oligo from IDT (whether ordering online, via email or via fax), use of the "+" prefix will serve to indicate the presence of an LNA base. This is also true when using IDT´s OligoAnalyzer for analysis of a sequence.

Depending on sequence context, insertion of an LNA base into a DNA oligo can increase the Tm by an average of 3-6^oC. IDT recommends that no more than 10 LNA bases be placed in a given oligo. Binding affinity (Tm) of LNA bases are LNA : LNA �2265; RNA �2265; DNA. Oligos with large numbers of LNA bases tend to form LNA:LNA hairpins or homodimers because of the very high Tm seen in LNA:LNA hybrids.

A variety of applications have been described where the increased Tm and/or nuclease stability of LNA bases improve oligonucleotide performance. A few of the exciting uses for LNAs with select references are provided below.

LNA Applications

1. Dual-Labeled Probes, Real-time PCR, Genotyping

   LNA bases can be placed at internal positions of linear DLPs, Molecular Beacons, or any other nucleic acid probe. The increased Tm of the LNA bases allows for shorter probes to be used; moreover, if the LNA bases are correctly positioned within the sequence they confer increased sensitivity to base mismatch (i.e., improve genotyping or SNP detection assays).

   Letertre, C., Perelle, S., Dilasser, F., Arar, K., and Fach, P. Evaluation of the performance of LNA and MGB probes in 5´-nuclease assays. Molecular and Cellular Probes, 17:307-311 (2003).

   Ugozzoli, L.A., Latorra. D., Pucket, R., Arar, K., and Hamby, K. Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Analytical Biochemistry, 324:143-152 (2004).

   
   
   
2. Antisense

   LNA bases can be used to make improved antisense chimeras in the same way that other 2´-modifications have been employed in the past, such as 2´-O-methyl RNA, MOE´s, etc. The use of LNAs offers improved Tm, increased resistance to cellular nucleases, and increased potency of the antisense oligo.

   Kurreck, J., Wyszko, E., Gillen, C., and Erdmann, V.A. Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Research, 30:1911-1918 (2002).

   Braasch, D.A., Liu, Y., and Corey, D.R. Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Research, 30:5160-5167 (2002).

   
   
   
3. siRNA (RNA Interference)

   A variety of chemical modifications are being used to improve stability and potency of siRNAs for use in RNA interference. While no single cocktail of modifications is widely accepted as "best", LNA, 2´-O-methyl RNA, and other RNA modifications are commonly used today in "second generation" siRNA designs.

   Braasch, D.A., Jensen, S., Liu, Y., Kaur, K., Arar, K., White, M.A., and Corey, D.R. RNA interference in mammalian cells by chemically-modified RNA. Biochemistry, 42:7967-7975 (2003).

   
   
   

Locked Nucleic Acids are licensed from Exiqon, http://www.exiqon.com/. For more information about LNA applications, Tm, and oligonucleotide design rules, see: http://lnatools.com.