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Niels Tolstrup et al. summarized by Ki-Roo Shin

OligoDesign: optimal design of LNA (locked nucleic acid) oligo-nucleotide capture probes for gene expression profiling. Niels Tolstrup et al. summarized by Ki-Roo Shin. Locked Nucleic Acids.

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Niels Tolstrup et al. summarized by Ki-Roo Shin

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  1. OligoDesign: optimal design of LNA (locked nucleic acid) oligo-nucleotide capture probes for gene expression profiling Niels Tolstrup et al. summarized by Ki-Roo Shin

  2. Locked Nucleic Acids • a novel class of nucleic acid analogues. LNA monomers are bicyclic compounds structurally similar to RNA nucleosides. • The term "Locked Nucleic Acid" has been coined to emphasize that the furanose ring conformation is restricted in LNA by a methylene linker that connects the 2'-O position to the 4'-C position (see figure). By convenience, all nucleic acids containing one or more LNA modifications are called LNA. • LNA oligomers obey Watson-Crick base pairing rules and hybridize to complementary oligo-nucleotides. LNA provides vastly improved hybridization performance when compared to DNA and other nucleic acid derivatives in a number of situations.

  3. Structure

  4. Thermal Stability of LNA • LNA/DNA or LNA/RNA duplexes have increased thermal stability compared with similar duplexes formed by DNA or RNA. LNA has the highest affinity towards complementary DNA and RNA ever reported. In general, the thermal stability of a LNA/DNA duplex is increased 3°C to 8°C per modified base in the oligonucleotide. • The LNA modification has been shown to increase the biological stability of nucleic acids. Fully modified LNA oligonucleotides are resistant towards most nucleases tested.

  5. Capture probes Sample preparation (mRNA) SNP analysis Mutation analysis Allele specific PCR Antisense Target Validation Hybridization probes Strand invasion In-situ hybridization Triple-helix forming oligos Nuclease protection assays Application

  6. Advantages • Affinity • LNA:LNA duplex formation constitutes the most stable Watson-Crick base pairing system yet developed. • LNA:LNA > LNA:RNA > RNA:RNA > RNA:DNA > DNA:DNA • Tm modulation • It is possible to fine-tune the placement of LNA bases to reach the desired Tm level without losing specificity. • Specificity • LNA enhances hybridization performance relative to native DNA and RNA, phosphorothiate orpeptide nucleic acid (PNA) probes. • LNA lowers experimental error rates due to better mismatch discrimination. • LNA provides more robust assay conditions. • LNA improves signal-to-noise ratio. • Simplicity & Design Flexibility

  7. OligoDesign Program • BLAST analysis • Prediction of duplex thermal stability and Tm equalization using LAN substitutions • Self-annealing • Prediction of the secondary structures

  8. Experimental Results • C. elegans microarray • 120 potential marker genes for a veriety of stress and toxicity processes and toxicologically relevant pathways, including drug metabolism, DNA damage-repair, apoptosis, stress response, membrane proteins and cell cycle regulators. • Design 50mer LNA substituted oligonuleotides • Two oligonucleotides showed high cross-hybridization signals to each other due to the high similarity (80%)

  9. References • Koshkin,A. et al. (1998) LNA: synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron, 54, 3607-3630 • Singh,S.K. et al. (1998) LNA: Synthesis and high-affinity nucleic acid recognition. Chem. Commun., 4, 455-456 • T.Koch (2003) LNA: a family of high affinity nucleic acid probes. J. Phys.: Condensed Matter, 15, S1861-S1871

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