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Page 8 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

Table 1. Comparison between natural phosphodiester ASOs and Double-stranded siRNA
ASO siRNA
Single-stranded Double-stranded
14-20 bases - linear deoxynucleotide 21-23 base pairs RNA
Flexible with ~ 1 nm width Rigid duplex ~ 2 nm diameter
Single-stranded nature requires full backbone modification with phosphorothioate (PS) Double-stranded nature ensures relative
linkages to protect from nucleases protection against nucleases
Design must retain ribose sugars moieties in the seed center sequence to allow RNase H Ribose sugars can be modified to a certain limit
activity with respect to Argonaute efficiency
Sugar modifications are tolerated only on wings of a gapmer Sugar modifications tolerated
Hydrophobic surfaces accessible for protein interactions allow binding to plasma proteins Little exposed hydrophobic surface since aromatic
such as albumin and increase blood circulation and biodistribution to tissues. bases are paired and buried in duplex.
Hydrophilic surface causes rapid kidney clearance.
ASO: antisense oligonucleotides; siRNA: small interfering RNA.

Many comprehensive excellent reviews have been dedicated to the intensive chemistry efforts performed on
ASO or siRNA derivatives, which have concerned their different components: backbone, sugar, and
base) [31-33] . The state-of-the-art will be briefly described here. The most successful chemical modifications
introduced so far on ASO and siRNA nucleotides are displayed in Figure 4. Because of their different
properties and mode of action, different chemical modifications have been selected after more than 20 years
of intensive research for each class of RNA drug.

The single-stranded nature of ASOs requires full phosphodiester backbone modification because of their
high exposure to exo- and endonucleases. Figure 4B displays the most successful modified backbone linkage
used so far, phosphorothioate (PS), in which a sulfur atom replaces one non-linking phosphate oxygen and
which partially protects against nucleolytic activity. In addition, phosphorothioate modification has been
shown to increase pharmacokinetics and cellular uptake [34-36] . Indeed, the first ASO drug introduced to the
market was the full 21 nt full phosphorothioate oligodeoxynucleotide Vitravene, which has the sequence 5’-
GCG TTT GCT CTT CTT CTT GCG-3’ targeting the CMV protein IE2 mRNA . Several other
[37]
phosphodiester linkage modifications also confer nuclease resistance, for instance, methylphosphonate,
phosphoramidate [Figure 4], and mesylphosphoramidate [38,39] .

It should be noted that each PS substitution introduces a chiral phosphate with two stereoisomers, the Sp
and Rp forms. The Sp diastereoisomers are more resistant to nucleases and should be preferred for a blocker
ASO. In addition, the Rp diastereoisomers display a higher binding affinity and induce more efficient RNase
H cleavage, which is more favorable for an mRNA degradation strategy. It has been shown that precise
localization of Rp and Sp PS links could optimize the desired function and increase selectivity by decreasing
[29]
off-target mismatch binding .

While phosphorothioate links confer improved relative nuclease resistance and metabolic stability, the
protection is not complete. Moreover, PS links decrease ASO and siRNA binding affinity to the
complementary mRNA. This can be corrected by introducing modifications to the ribose moiety.
Modifications on the 2’ carbon, such as 2’-fluoro, 2’-O-methyl or 2’-O-metoxyethyl are extensively used for
both ASOs and siRNAs [Figure 4C]. Another way to increase nuclease resistance is by introducing
constraints in the ribose ring, such as in the bicyclic locked nucleic acids (LNA) or in the constrained ethyl
locked nucleic acids (cet-LNA).