Key featuresShow Hide
- Locked nucleic acid backbone modification of oligos confers enhanced duplex stability and specificity
- LNA oligos are synthesized using routine reagents, and the stability of LNA to base results in compatibility with the most common cleavage and deprotection strategies.
- LNA has similar solubility, handling and purification properties to DNA
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In the cell, double-stranded DNA exists as a B-form helix, while double-stranded RNA adopts an A-form helical structure. This arises from the differences in the preferred conformations of the sugar ring of deoxyribose and ribose. In DNA, the furanose ring of deoxyribose predominately exists in the C2'-endo conformation resulting in the B-form helix. In RNA, the furanose ring exists in C3'-endo confirmation due to the presence of the 2'-OH resulting in the A-form helix.
In 1998, laboratories in Japan and Denmark first described the synthesis and properties of a novel series of nucleic acid analogues called Locked Nucleic Acids (LNA) (1). These are locked in the C3'-endo conformation by means of a methylene bridge that connects the 2’ oxygen atom to the 4’ carbon atom. This bridging restricts the conformational flexibility and results in the pre-organisation of the sugar in an RNA-like form.
Physical studies of LNA-containing oligonucleotides hybridised to DNA or RNA revealed some remarkable properties. The melting temperatures (Tm) were dramatically increased and, most importantly, not at the expense of mismatch discrimination. The specificity of LNA for its perfectly matched complement is significantly higher. An increase in Tm of as much as 41 °C for a full-LNA:DNA duplex relative to the corresponding DNA:DNA duplex has been reported. In general, an increase in Tm of about 3-8 °C per LNA modification is observed. The structures of LNA containing oligonucleotides hybridised to both DNA and RNA have been determined in solution by NMR. These show that the LNA residues induce a conformational change in the surrounding DNA causing it to adopt a similar C3'-endo conformation. This leads to an increased local organisation of the phosphate backbone, enhancing the strength of base stacking interactions, leading to increased duplex stability observed as increased Tm values.
The binding of LNA to double-stranded DNA under physiological conditions has also been observed. This can occur by triplex formation or by strand invasion mechanisms and raises the prospect of being able to use LNA to recognise double-stranded DNA within living cells.
Full-LNA is nuclease resistant, this can also be achieved in chimeras by the incorporation of phosphorothioate linkages in the DNA sections of the molecule.
Unlike some other modifications, such as morpholino oligonucleotides or PNA, oligonucleotides can be made routinely by automated synthesis by phosphoramidite chemistry with no additional reagents with only minor modifications to synthesis cycles. Longer coupling time compared to DNA additions are required in addition to a prolong the oxidation step.
The use of standard nucleobase protection and the stability of LNA to base results in compatibility to the most common cleavage and deprotection strategies. Although there is an increased hydrophobicity, LNA has similar solubility and handling properties established purification methods can be utilised. This simplicity of preparation combined with its outstanding hybridisation properties makes LNA an extremely attractive choice in DNA modification.
In addition to offering the LNA phosphoramidites, Biosearch Technologies also manufacturers a range of LNA CPGs in different pore sizes, in bulk and in columns.
- (a) Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2 ‘-O,4 ‘-C-methyleneribonucleosides, S. Obika, D. Nanbu, Y. Hari, J. Andoh, K. Morio, T. Doi, and T. Imanishi, Tetrahedron Lett., 39, 5401-5404, 1998. (b) LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition, A.A. Koshkin, S.K. Singh, P. Nielsen, V.K. Rajwanshi, R. Kumar, M. Meldgaard, C.E. Olsen, and J. Wengel, Tetrahedron, 54, 3607-3630, 1998.