Custom LNA Oligonucleotides

LNA oligonucleotides exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. For each incorporated LNA monomer, the melting temperature (T_m) of the duplex increases by 2–8°C (see figure Replace DNA with LNA for higher melting temperature). In addition, LNA oligonucleotides can be made shorter than traditional DNA or RNA oligonucleotides and still retain a high T_m. This is important when the oligonucleotide is used to detect small or highly similar targets.

Since LNA oligonucleotides typically consist of a mixture of LNA and DNA or RNA, it is possible to optimize the sensitivity and specificity by varying the LNA content of the oligonucleotide. Incorporation of LNA into oligonucleotides has been shown to improve sensitivity and specificity for many hybridization-based technologies including PCR, microarrays and in situ hybridization (ISH).

T_m normalization enables robust detection, regardless of GC content. The T_m of a nucleotide duplex can be controlled by varying the LNA content. This feature can be used to normalize the T_m across a population of short sequences with varying GC content. For AT-rich nucleotides, which give low melting temperatures, more LNA is incorporated into the LNA oligonucleotide to raise the T_m of the duplex. This enables the design of LNA oligonucleotides with a narrow T_m range, which is beneficial in many research applications such as microarrays, PCR and other applications in which sensitive and specific binding to many different targets must occur under the same conditions simultaneously. The power of T_m normalization is demonstrated by the comparison of DNA and LNA probes for detection of miRNA targets with a range of CG content (see figure The power of T_m normalization).

Use the guidelines below when designing your own Custom LNA Oligonucleotides:

• LNA will bind very tightly to other LNA residues. Avoid self-complementarity and cross-hybridization to other LNA-containing oligonucleotides
• Keep the GC content between 30–60%
• Avoid stretches of more than 4 LNA bases, except when very short (9–10 nucleotides) oligonucleotides are designed
• Avoid stretches of 3 or more Gs or Cs
• For novel applications, design guidelines may have to be established empirically

LNA oligonucleotides can be successfully used in a wide range of applications, including:

• miRNA research
• Small RNA research
• SNP genotyping
• mRNA antisense oligonucleotides
• Allele-specific PCR
• RNAi
• DNAzymes
• Fluorescence polarization probes
• Molecular beacons
• Microarray gene expression profiling
• Gene repair/exon skipping
• Splice variant detection
• Comparative genome hybridization (CGH)