Phosphorothioates and Chimeric Oligos
While unmodified oligodeoxynucleotides can display some antisense activity, they are subject to rapid degradation by nucleases and are therefore of limited utility. The simplest and most widely used nuclease-resistant chemistry available for antisense applications is the phosphorothioate (PS) modification. In phosphorothioates, a sulfur atom replaces a non-bridging oxygen in the oligo phosphate backbone. In the IDT ordering system, an asterisk indicates the presence of a phosphorothioate internucleoside linkage. PS oligos can show greater non-specific protein binding than unmodified phosphodiester (PO) oligos, which can cause toxicity or other artifacts when present at high concentrations. These problems seem to be worst with PS-DNA and PS-DNA/LNA chimeras. Non-specific protein binding of PS oligos can be minimized by making the oligonucleotide as short as possible (thereby reducing PS content) or using chimeric designs with 2'-O-methyl RNA.
LNA, 2′-O-Methyl RNA, and 5-Methyl dC
State-of-the-art antisense design employs chimeras having 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. IDT recommends that all antisense oligos receive HPLC purification and that oligos undergo a Na+ salt exchange before use in cells or live animals to ensure that salts used in purification are removed.
|Examples of RNase H Active Antisense Oligos |
|5′ T*C*C*T*G*C*G*A*A*A*T*G*T*C*C*A*T*C*G*T 3′ ||DNA, All PS |
|5′ T*C*C*TGCGAAATGTCCAT*C*G*T 3′ ||DNA, PS/PO chimera |
|5′ mU*mC*mC*mU*mG*C*G*A*A*A*T*G*T*C*C*mA*mU*mC*mG*mU 3′ ||2′OMe/DNA chimera, All PS |
|5' +T+C+C+T+GC*G*A*A*A*T*G*T*C*C+A+T+C+G+T/3Phos/3′ ||LNA/DNA chimera, PS/PO chimera, 3′-phos |
* = Phosphorothioate Bonds
mN = 2′-O-Me RNA base
+N = LNA base