Locked nucleic acid (LNA) forms DNA- or RNA-heteroduplexes with exceptional high thermal stability, and has the potential to improve current in vitro DNA diagnostic technologies. Jan Skouv, Nana Jacobsen and Mogens Havsteen Jakobsen report the latest research and what is likely to be the impact on today's and tommorow's technologies.
DNA oligonucleotides have found widespread use in molecular biology research and the rapidly expanding field of DNA diagnostics. One just has to think of the application of DNA oligos as gene specific probes, as primers for PCR, as target molecules on DNA-arrays and a number of other highly specific in vitro diagnostic applications to realise that even modest improvements in their performance are bound to have a large impact on present day technologies and likely to inspire to new diagnostic technologies.
Locked nucleic acid (LNA) is a novel class of DNA analogues that possesses some extraordinary features making it a prime candidate for in vitro DNA diagnostics and in particular for detection of single base nucleotide polymorphisms (SNPs). The LNA monomers are bicyclic compounds structurally very similar to DNA-monomers, see Fig. 1.
LNA share most of the chemical properties of DNA and RNA, it is water-soluble, can be separated by agar gel electrophoreses, ethanol precipitated etc. However, introduction of LNA monomers into either DNA or RNA oligos results in unprecedented high thermal stability of duplexes with complementary DNA or RNA, while, at the same time obeying the Watson-Crick base-pairing rules (1). In general, the thermal stability of heteroduplexes is increased 38°C pr. LNA-monomer in the duplex. Thus a heteroduplex between a DNA and a complementary 9-mer LNA-oligo usually have a melting temperature (Tm) well above 60°C in hybridisation buffer containing 120 mM Na+ (1 X SSC contains 165 mM Na+).
To the best of our knowledge LNA has the highest affinity and specificity towards complementary DNA or RNA ever to be reported (ref 1). Since LNA-oligos are synthesised by phosphoramidite chemistry, chimera oligos, containing both LNA and DNA (or RNA) monomers can be synthesised. This allows one to design mixed LNA/DNA(RNA) oligos with a predefined Tm.
LNA in molecular diagnostics
LNA's extraordinary hybridisation properties together with Exiqon's proprietary photochemical method for covalent attachment of oligonucleotides (2, 3) have been developed to form the basis for highly efficient microtiter plate based assays for detection of SNPs. We have successfully applied LNA and our coupling technology to develop assays that detect the Apolipoprotein B R3500Q mutation (4), Leiden factor V mutation (5) and the Apolipoprotein E genotype (6) in human samples. Because of LNA's extraordinarily high Tm very short oligos can be used as specific catching oligos in these assays.
As illustrated in Table 1, such short catching LNA-oligos are characteristic of having large differences in the Tm (DTm) of complementary versus single base-mismatching duplexes. It is in particular interesting to note that the DTms of LNA are significant higher than the DTms of DNA with similar sequence (see Table 1). This feature of LNA results in hybridisations that are highly discriminative towards single base differences. Using an 8-mer as catching oligo we have observed more than a 100-fold difference in hybridisation signal between matching PCR fragment and a similar PCR fragment containing a single mismatch.
A particularly interesting application of LNA is in the development of nucleotide micro-arrays for the simultaneous detection multiple SNPs. To achieve an adequate performance such arrays requires probes of the highest specificity. Together with Exiqon's photo-chemical immobilisation method LNA forms the basis for the EURAY, a bioarray system, that will facilitate simultaneous analysis of a large number of targets thus providing high throughput capabilities of DNA based diagnostics.
Finally LNA-oligos can be designed that are substrate for a number of polymerases including Taq polymerase. Fully modified LNA oligos do not function as primers in PCR, however, primers with a single 3' LNA-modified base do. Interestingly, such 3' LNA-modified PCR primers seem to be superior relative to similar DNA primers to distinguish between two alleles that only differ at one nucleotide position (allele specific PCR).
In conclusion, LNA is a novel class of nucleotide analogues that forms duplexes with complementary DNA and RNA with highly increased thermal stability and generally improved selectivity. Our results show that LNA is able effectively to detect single nucleotide polymorphisms and that LNA-based assays in some respects are superior to similar DNA analysis methods.
ENQUIRY No 62
Jan Skouv , Nana Jacobsen and Mogens Havsteen Jakobsen are with Exiqon A/S, Vedbaek, Denmark. www.exiqon.com
References: 1. Koshkin, A K, Singh, S K, Nielsen, P et al. Tetrahedron 1998; 54: 3607-30. 2. Koch, T, Jacobsen, N, Fensholdt, J, Boas, U, Fenger, M, and Jacobsen, M H Bioconjugate Chemistry 2000:11, 474-483. 3. www.exiqon.com 4. Tybjaerg-Hansen A, Humphries SE. Atherosclerosis 1992; 96:91-107. 5. Ørum, H, Jakobsen, M H, Koch, T, Vuust, J, and Borre, M B Clinical Chemistry 1999, 45 (10), 1898-1905. 6. Mahley, R T Science 1988; 240: 622-630.