Duplex Stability and Nuclease Resistance

Applications & Recommended Modifications

The following guideline represents the wide application of modified base use for almost all oligo and probe design and use. Gene Link technical service offers advice on oligo design and use of modifications based on application.

Application	Recommended Modifications
Antisense Gene Target	Oligonucleotides containing 2'-OMe-nucleotides (2'-OMe-RNA) forms more stable hybrids with complementary RNA strands than equivalent DNA and RNA sequences. Phosphorothioate linkages confer oligonucleotides resistance to nuclease degradation 3′ Cholesterol modification helps in cellular uptake.
RNA Interference (siRNA)	Alternating 2′-F bases and 2′OMe bases siRNA enhances duplex stability and are more resistant to RNase degradation. Phosphorothioate linkages confer oligonucleotides resistance to nuclease degradation. Incorporate 2′-F bases, 5-me dC or 2-amino dA preferentially at the 5′ end of the sense strand to block incorporation of the sense strand in to the RISC. 2′F U and C substituted siRNA are more resistant to RNase degradation. 3′ Cholesterol modification helps in cellular uptake.
Real-Time PCR probes, QPCR	Appropriately substituted 2′Amino dA and 5�Me dC bases imparts greater specificity with higher Tm. Substituting 4-6 bases increases the Tm by 6-10 degrees. These probes enhance duplex stability and thus shorter probes can be synthesized. All types of fluorescent dyes and backbone modifications can be performed. 5 methyl dC behave similar to LNA bases in imparting duplex stability.
SNP Genotyping, Allelic Discrimination	Appropriately substituted 2-Amino dA and 5-Me dC bases imparts greater specificity with higher Tm. Substituting 4-6 bases increases the Tm by 6-10 degrees. These primers and probes enhance duplex stability and thus shorter primers and probes can be synthesized. All types of fluorescent dyes and backbone modifications can be performed. 5 methyl C behave similar to LNA bases in imparting duplex stability.
Fluorescent in situ Hybridization Probes (FISH)	Design multiple 24 to 30mer probes. Avoid stretches of more than 3 G or C bases. To impart exonuclease resistance substitute 3-4 bases at the 5′ and 3′ end with 2′F bases. The 2′ F bases imparts resistance to exonuclease degradation and increases duplex stability by 4-6 degrees. Several internal bases can be substituted with 5me dC and 2 Amino dA to further increase duplex stability. 5 methyl C behave similar to LNA bases in imparting duplex stability. Affinity ligands such as Digoxigenin or Biotin or fluorescent dye e.g Cy3, Cy5 or any other can be labeled at the 3′ and 5′ end. Multiple internal sites can also be labeled with affinity ligands or fluorescent dyes to increase sensitivity. Multiple dye sites should be spaced apart by 10 or more bases. The above guidelines are for all initial FISH probe design. Design rules may have to be established empirically for very specific or novel assay settings, but following the above recommendations will provide a good start.
PCR Amplification Primers	To impart exonuclease resistance substitute 3-4 bases at the 5′ and 3′ end with 2′F bases. The 2′ F bases imparts resistance to exonuclease degradation and increases duplex stability by 4-6 degrees. Several internal bases can be substituted with 5me dC and 2 Amino dA to further increase duplex stability. 5 methyl C behave similar to LNA bases in imparting duplex stability.