Locked nucleic acid
A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such oligomers are synthesized chemically and are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides.[1]
LNA was independently synthesized by the group of Jesper Wengel[2] in 1998, soon after the first synthesis by the group of Takeshi Imanishi[3] in 1997. The exclusive rights to the LNA technology were secured in 1997 by Exiqon A/S, a Danish biotech company.[4]
LNA nucleotides are used to increase the sensitivity and specificity of expression in DNA microarrays, FISH probes, real-time PCR probes and other molecular biology techniques based on oligonucleotides. For the in situ detection of miRNA the use of LNA is currently (2005) the only efficient method. A triplet of LNA nucleotides surrounding a single-base mismatch site maximizes LNA probe specificity unless the probe contains the guanine base of G-T mismatch.[5]
Using LNA based oligonucleotides therapeutically is an emerging field in biotechnology. The Danish pharmaceutical company Santaris Pharma a/s owns the sole rights to therapeutic uses of LNA technology,[6] and is now developing a new, LNA based, hepatitis C drug called miravirsen, targeting miR-122, which is in Phase II clinical testing as of late 2010.[7]
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[edit] Benefits of the LNA technology
Some of the benefits of using LNA include:
- Ideal for the detection of short RNA and DNA targets
- Increases the thermal stability of duplexes
- Capable of single nucleotide discrimination
- Resistant to exo- and endonucleases resulting in high stability in vivo and in vitro applications
- Increased target specificity
- Facilitates Tm normalization
- Strand invasion properties enables detection of “hard to access” samples
- Compatible with standard enzymatic processes[8]
[edit] Applications of the LNA technology
Some proven applications of LNA include:
- Allele-specific PCR: allows for the design of shorter primers, without compromising binding specificity [9]
- Microarray gene expression profiling: provides increased sensitivity and selectivity with smaller amounts of substrates[10]
- Small RNA research
- SNP genotyping
- mRNA antisense oligonucleotides
- RNAi
- DNAzymes
- Fluorescence Polarization probes
- Molecular Beacons
- Gene repair/exon skipping
- Splice variant detection
- Comparative genome hybridization (GCH)[11]
Other therapeutic and diagnostic applications of LNA technology are in development.[11]
[edit] References
- ^ Kaur, H; Arora, A; Wengel, J; Maiti, S; Arora, A.; Wengel, J.; Maiti, S. (2006). "Thermodynamic, Counterion, and Hydration Effects for the Incorporation of Locked Nucleic Acid Nucleotides into DNA Duplexes". Biochemistry 45 (23): 7347–55. doi:10.1021/bi060307w. PMID 16752924.
- ^ Alexei A. Koshkin; Sanjay K. Singh, Poul Nielsen, Vivek K. Rajwanshi, Ravindra Kumar, Michael Meldgaard, Carl Erik Olsen, Jesper Wengel (1998). "LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition". Tetrahedron 54 (14): 3607–30. doi:10.1016/S0040-4020(98)00094-5.
- ^ Satoshi Obika; Daishu Nanbu, Yoshiyuki Hari, Ken-ichiro Morio, Yasuko In, Toshimasa Ishida, Takeshi Imanishi (1997). "Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3'-endo sugar puckering". Tetrahedron Lett. 38 (50): 8735–8. doi:10.1016/S0040-4039(97)10322-7.
- ^ http://www.exiqon.com/history
- ^ You Y.; Moreira B.G.; Behlke M.A. and Owczarzy R. (2006). "Design of LNA probes that improve mismatch discrimination". Nucleic Acids Res. 34 (8): e60. doi:10.1093/nar/gkl175. PMC 1456327. PMID 16670427. http://nar.oxfordjournals.org/cgi/reprint/34/8/e60?ijkey=IqLeijzpwQHCzit&keytype=ref.
- ^ "Developing LNA technology for new-generation cancer drugs" (PDF). SP2 Magazine. March. http://www.ebiotrade.com/emgzf/spII200604/16-17.pdf.
- ^ Franciscus, Alan (2010). "Hepatitis C Treatments in Current Development". HCV Advocate. http://www.hcvadvocate.org/hepatitis/hepC/HCVDrugs.html.
- ^ Locked Nucleic Acid Technology
- ^ Bonetta, Laura (2005). "Prime time for real-time PCR". Nat. Methods 2 (4): 305–312. doi:10.1038/nmeth0405-305.
- ^ Roberts, Peter (2006). "MicroRNA expression profiling on arrays enhanced with locked nucleic acids". Nat. Methods 3 (4). doi:10.1038/nmeth869. http://www.nature.com/nmeth/journal/v3/n4/full/nmeth869.html.
- ^ a b http://www.exiqon.com/custom-lna-oligos
[edit] External links
- LNA Oligo Tools and Design Guidelines
- LNA Oligo melting temperature including mismatches
- LNA Summary
- Vester B, Wengel J (October 2004). "LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA". Biochemistry 43 (42): 13233–41. doi:10.1021/bi0485732. PMID 15491130.
- Petersen M, Wengel J (February 2003). "LNA: a versatile tool for therapeutics and genomics". Trends Biotechnol. 21 (2): 74–81. doi:10.1016/S0167-7799(02)00038-0. PMID 12573856. http://linkinghub.elsevier.com/retrieve/pii/S0167779902000380.
- Ng PS, Bergstrom DE (January 2005). "Alternative nucleic acid analogues for programmable assembly: hybridization of LNA to PNA". Nano Lett. 5 (1): 107–11. doi:10.1021/nl048246f. PMID 15792422. http://pubs.acs.org/cgi-bin/sample.cgi/nalefd/2005/5/i01/abs/nl048246f.html.
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