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Locked nucleic acid

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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 and hybridize with DNA or RNA according to Watson-Crick base-pairing rules. 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. LNA was independently synthesized by the group of Jesper Wengel in 1998, soon after the first synthesis by the group of Takeshi Imanishi in 1997. The exclusive rights to the LNA technology were secured in 1997 by Exiqon A/S, a Danish biotech company.

Contents

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.

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, 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.

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

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

    • 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
  • Microarray gene expression profiling: provides increased sensitivity and selectivity with smaller amounts of substrates
  • Small RNA research
  • SNP genotyping
  • mRNA antisense oligonucleotides
  • RNAi
  • Deoxyribozymes
  • Fluorescence Polarization probes
  • Molecular Beacons
  • Gene repair/exon skipping
  • Splice variant detection
  • Comparative genome hybridization (GCH)

    • Other therapeutic and diagnostic applications of LNA technology are in development. A cooperation between University of Southern Denmark and Decan University are focusing on using LNA to diagnose cancer-stem cells, 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.

    References

    Locked nucleic acid Wikipedia
    (Text) CC BY-SA