• ODNs must be nuclease resistant before and during residence in cells,
  ODNs must have the ability to cross the cellular membrane with some level of efficiency,
• ODNs must demonstrate high binding affinity and specificity for the target sequence.

Resistance towards nucleases
Nuclease resistance is fairly easy to achieve by :

• The modification of the normal phosphodiester backbone (e.g., phosphorothioates, methylphosphonates)
• The incorporation of 2’-OMe-nucleotides (2’-OMe-RNA)
• The use of Locked Nucleic Acids (LNA^®)
• The use of a 3’-terminal cap (e.g., 3’-aminopropyl modification or by using a 3’-3’ terminal linkage)

Ability to cross the cell membrane
Transport of oligonucleotides into cells is a problem routinely faced by antisense researchers. Improved transport through the cellular membrane can be achieved by :

• The use of a carrier molecule linked to the antisense oligonucleotide (e.g., cholesterol),
• The use of tranfection reagents • Backbone modification to more lipophilic linkages (methylphosphonate),
• The incorporation of modified monomers (5-(1-propynyl)-2’-deoxy-Uridine (pdU) and 5-(1-propynyl)-2’-deoxyCytidine (pdC).

Binding affinity and specificity
Increasing the affinity and specificity of an oligonucleotide has been more difficult to achieve since this necessitates modifying the natural bases which are already almost perfectly set up for optimal hydrogen bonding.

To achieve this, it has been described that the recommended modifications are:

• Oligonucleotides containing 2’-OMe-nucleotides (2’-OMe-RNA) form more stable hybrids with complementary RNA strands than equivalent DNA and RNA sequences,
• Phosphorothioate linkages confer to the oligonucleotides a higher binding affinity,
• C-5 methylated pyrimidine deoxy-nucleosides are known to form more stable duplexes and triplexes than their corresponding pyrimidine derivatives,
• 5-(1-propynyl)-2’-deoxy-Uridine (pdU) and 5-(1-propynyl)-2’-deoxyCytidine (pdC) monomers demonstrated that both substitutions enhanced duplex stability.