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Locked nucleic acid ­ high affinity nucleic acid analogues

1st April 2013


Locked nucleic acid (LNA) forms exceptionally thermostabile duplexes with complementary DNA and RNA and has the potential to improve current in vitro DNA diagnostic technologies and therapeutic drug development. Report by Jan Skouv, Henrik Ørum and Mogens Havsteen Jakobsen.

DNA oligonucleotides have found widespread use in molecular biology research, in the rapidly expanding field of DNA diagnostics and in the development of therapeutic drug candidates.

There are, however, a number of shortcomings with DNA oligos that in many instances complicate their use, most notable their low stability in biological fluids, and their relative modest affinity for complementary nucleic acids. To solve these problems considerable efforts have been invested in developing DNA analogues with improved hybridisation characteristics and biostability.

Locked nucleic acid (LNA) is a novel class of DNA analogues that appears to meet these requirements. LNA was invented by Professor Jesper Wengel, University of Copenhagen and Dr Poul Nielsen, Odense University in 1996 and was soon after licensed exclusively to Exiqon A/S.

The LNA monomer (Fig. 1) is a bicyclic compound in which the 2' and 4' position of the furanose ring are linked by an O-methylene (oxy-LNA), S-methylene (thio-LNA) or NH-methylene moiety (amino-LNA). This linkage restricts the conformational freedom of the furanose ring and hence the name alocked nucleic acid'.

Introduction of LNA monomers into DNA or RNA oligomers dramatically increases their affinity for complementary DNA or RNA (measured as the thermal stability of the duplexes, Tm). For instance, a DNA duplex (5'-GTGATATGC-3' : 5'-GCATATCAC-3') have a Tm of 28oC in 100 mM NaCl, whereas the Tm of the similar LNA:DNA heteroduplex is 64oC. In general, increases in affinity ranges from 3 to 8oC per LNA monomer depending on the sequence and number of LNA monomers in the oligo. In all cases analysed, both fully and partly LNA modified oligomers have been found to obey the Watson-Crick hydrogen bonding rules and to display excellent target specificity.

In most other aspects LNA behaves very much like DNA. It is water-soluble; the thermo-stability of its duplexes with complementary nucleic acids can be modulated by the ionic strength of the hybridisation solution; it functions as a substrate for a variety of nucleic acid enzymes such as polymerases, terminal transferases and kinases; and it lends itself to many of the routine methods that are applied to DNA, such as ethanol-precipitation, gelelectrophoresis, mass spectroscopy, etc.

Perhaps as important as its physical properties, it is a fact that LNA-oligos are made by the phosphoramidite chemistry using standard DNA synthesisers. Thus, chimerical oligos containing both LNA and DNA (or RNA) monomers as well as fully modified LNA oligos are easily synthesised. The flexibility of the phosphoramidite synthesis approach furthermore facilitates the easy synthesis of LNAs carrying all standard linkers, fluorophores and reporter-groups.

LNA in molecular diagnostics

The development of high affinity/high specificity probes is central to the successful implementation of DNA technology based products in the routine clinical laboratories. In lengthy diagnostic procedures, for instance, the probes need to have high affinity to secure adequate sensitivity of the test and good specificity to avoid false positive results. Thus, with its hybridisation characteristics LNA seems poised for success.

Applications for LNA may range from simply replacing DNA in many of its current uses to the development of novel technologies that cannot be made with DNA.

A particularly interesting application of LNA is in the development of DNA microchips, which requires probes of the highest specificity. Such microchips facilitate simultaneous analysis of a large number of targets thus providing high throughput capabilities at competitive prices.

To exploit this potential, Exiqon has entered into a collaborative agreement with the Micro Electronic Center (MIC) at the Danish Technical University. The goal is to develop a bio-array platform, termed EURAY, that can be used for both DNA and protein based diagnostics. Exiqon will bring its LNA and its photochemical coating technology to the collaboration. The latter of these technologies enables the covalent attachment of macromolecules to solid surfaces by light and is suited for laser-based miniaturisation. MIC will bring its expertise within microfluidics, detection systems and system integration to the collaboration.

Initially, the focus will be on diagnosing genetically inherited diseases. Clinical testing of the microchips will be performed at Hvidovre University Hospital, which is also engaged in the EURAY initiative. LNA also has a significant potential in the development of novel therapeutic drugs. In particular, the extraordinary high Tm of LNA and its biological stability make it ideal for antisense drug development.

Conclusion

LNA is a high affinity, highly biostable DNA analogue. These features, combined with LNA's close physical relatedness to DNA, make it highly attractive as a tool in biological research and DNA diagnostics and in the development of therapeutic drugs. It seems likely that many of the current technologies that uses DNA, or other moderate affinity analogues, can be improved significantly by switching to LNA. Also, it seems likely that LNA will facilitate the development of novel technologies that cannot be achieved with standard DNA.

Enquiry No 28

Jan Skouv, PhD, is manager of molecular biology, Henrik Ørum, PhD, is member of the scientific advisory committee and the board of directors, and Mogens Havsteen Jakobsen, PhD, is vice president and director of research and development, at Exiqon A/S in Vedbaek, Denmark.





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