Locked nucleic acid

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Chemical structure of an LNA monomer an additional bridge bonds the 2' oxygen and the 4' carbon of the pentose

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] [2] LNA was independently synthesized by the group of Jesper Wengel[3] in 1998, soon after the first synthesis by the group of Takeshi Imanishi[4] in 1997. The exclusive rights to the LNA technology were secured in 1997 by Exiqon A/S, a Danish biotech company.[5]

Conformational structure of an LNA monomer β-D configuration

LNA nucleotides are used to increase the sensitivity and specificity of expression in DNA microarrays, FISH probes, quantitative 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.[2][6]

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,[7] 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.[8]

An LNA-specific molecular dynamics (MD) force field has been created [9] which is able to accurately model LNA MD.

Benefits of the LNA technology[edit]

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[10]

Applications of the LNA technology[edit]

Some proven applications of LNA include:

  • Allele-specific PCR: allows for the design of shorter primers, without compromising binding specificity [11]
  • Microarray gene expression profiling: provides increased sensitivity and selectivity with smaller amounts of substrates[12]
  • 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)[13]

Other therapeutic and diagnostic applications of LNA technology are in development. A cooperation between Southern University of Denmark and Decan University are focusing on using LNA to diagnose cancer-stamcells, which will be a major leap forward to avoid cancer-patients having relapse. The team filed patent in February 2014 and will continue with trials on rodents in late 2014.[13]

Notes and references[edit]

  1. ^ Kaur, H; 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. 
  2. ^ a b Owczarzy R.; You Y.; Groth C.L.; Tataurov A.V. (2011). "Stability and mismatch discrimination of locked nucleic acid-DNA duplexes.". Biochem. 50 (43): 9352–9367. doi:10.1021/bi200904e. PMC 3201676. PMID 21928795. 
  3. ^ 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. 
  4. ^ 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. 
  5. ^ History
  6. ^ 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. 
  7. ^ "Developing LNA technology for new-generation cancer drugs" (PDF). SP2 Magazine. March 2006. 
  8. ^ Franciscus, Alan (2010). "Hepatitis C Treatments in Current Development". HCV Advocate. 
  9. ^ David E. Condon; Ilyas Yildirim; Scott D. Kennedy; Brendan C. Mort, Ryszard Kierzek; Douglas H. Turner (December 2013). "Optimization of an AMBER Force Field for the Artificial Nucleic Acid, LNA, and Benchmarking with NMR of L(CAAU)". J. Phys. Chem. B 118 (5): 1216–1228. 
  10. ^ Locked Nucleic Acid Technology
  11. ^ Bonetta, Laura (2005). "Prime time for real-time PCR". Nat. Methods 2 (4): 305–312. doi:10.1038/nmeth0405-305. 
  12. ^ Roberts, Peter (2006). "MicroRNA expression profiling on arrays enhanced with locked nucleic acids". Nat. Methods 3 (4). doi:10.1038/nmeth869. 
  13. ^ a b Custom Locked Nucleic Acid (LNA™) oligonucleotides

External links[edit]