LNA, 2′-O-methyl RNA, and 5-methyl dC
State-of-the-art antisense design employs chimeras with both DNA and modified-RNA bases. The use of modified RNA, such as 2′-O-methyl (2′OMe) RNA or LNAs (Exiqon) in chimeric antisense designs, increases both nuclease stability and affinity (Tm) of the antisense oligo to the target mRNA [3–5]. These modifications, however, do not activate RNase H cleavage. The preferred antisense design incorporates 2′-O-modified RNA or LNA in chimeric antisense oligos that retain an RNase H activating domain of DNA (or phosphorothioate DNA). As LNA bases confer significant nuclease resistance, we recommend phosphorothioate modification of only the DNA gap, leaving the LNA flanks as phosphodiester linkages in chimeric LNA antisense oligos. For synthesis reasons, a 3′-phosphate is preferred when an LNA base is at the 3′ end.
It can also be beneficial to substitute 5-Methyl-dC for dC in the context of CpG motifs. Substitution of 5-Methyl dC for dC will slightly increase the Tm of the antisense oligo. Use of 5-Methyl dC in CpG motifs can also reduce the chance of adverse immune responses in vivo. We recommend HPLC purification and Na+ salt exchange for all antisense oligos before use in cells or live animals to ensure that salts used in purification are removed.