| Mesyl phosphoramidate (MsPA or µ) is a chemical modification that is useful in the design of therapeutic antisense oligonucleotides (ASOs). The Stetsenko lab (Patutina et al., 2020) reported that the MsPA linkage is an alternative to phosphorothioate (PS) for RNase H1-mediated knockdown of microRNA targets and for modulating mRNA splicing. In the MsPA linkage, one of the non-bridging oxygen atoms in the phosphodiester linkage is replaced with a methanesulfonylamido group (Figure 1), unlike in phosphoramidate and alkylphosphonate linkages, thereby retaining a negative charge on the phosphate backbone. However, it lacks the negatively charged sulfur atom that is the primary pharmacophore for ASO-protein interactions. The MsPA linkage can be introduced at any position within oligonucleotides and combined with other backbone and sugar modifications during the oxidation cycle via a Staudinger reaction between mesyl azide and the phosphite intermediate formed after phosphoramidite coupling. |  Figure 1: MsPA or µ, µ-ODNB: Oligodeoxynucleotide mesyl phophor-amidate, negatively charged. |
According to Su et al. (2019), the PN backbone is less toxic than the phosphorothioate backbone. According to Patutina et al. (2020), synthetic oligonucleotides modified with mesyl phosphoramidates (or µ-) show high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro.
One of the biggest challenges in nucleic acid therapeutics is that nucleases in blood and cells rapidly degrade natural DNA/RNA. MsPA linkages are exceptionally resistant to nucleases and more stable than phosphorothioate (PS) linkages, allowing drugs to remain intact in the body for extended periods.
MsPA is currently being investigated for use in "Gapmer" ASOs. In these designs, MsPA linkages are often strategically placed in the "gap" region, the center of the DNA strand, to encourage RNase H activity while reducing overall toxicity resulting from a complete phosphorothioate backbone.
For gapmers to work, they must trigger RNase H to degrade the disease-causing RNA target. Many chemical modifications, including 2'-O-methyl or Morpholinos, prevent this enzyme from working. MsPA is one of the few backbone modifications that recruit RNase H efficiently. Because MsPA mimics the geometry and charge of the natural phosphodiester bond better than many other bulky modifications, it allows the RNase H enzyme to recognize and cleave the target RNA strand.
Modifications that recruit RNase H1, such as PS, mesylphosphoramide, or 5′-O-methyl-phosphonate for irreversible RNA degradation, may significantly increase not only nuclease resistance, but also reaction turnover. However, the placement of modifications within the catalytic core of DNAzymes need careful consideration, as even minor structural changes can disrupt the active conformation and dramatically reduce catalytic activity.
A therapeutic oligonucleotide must hybridize tightly to its target RNA to be effective. MsPA-modified oligonucleotides form stable duplexes with RNA targets. Phosphorothioate (PS) modifications typically lower the duplex melting temperature (Tm) slightly, weakening binding. In contrast, MsPA linkages maintain or even improve binding affinity compared to PS, often performing nearly as well as natural DNA.
Toxicity is the primary limiting factor for current therapeutic phosphor-thioates, which can cause immune reactions and liver damage. MsPA oligonucleotides have shown a better safety profile in animal studies, with lower liver enzyme elevations (ALT/AST) than PS. MsPA oligonucleotides exhibit less nonspecific binding to cellular proteins, thereby reducing side effects associated with PS-modified drugs.
Unlike the neutral backbones of PMOs or PNAs, MsPAs retain a negative charge, maintaining their water solubility and allowing them to be taken up by cells via pathways similar to those for natural DNA. The mesyl group is chemically stable under the standard conditions used to synthesize and deprotect oligonucleotides, making it compatible with standard manufacturing processes that often utilize the Staudinger reaction.
In 2019, Su et al. evaluated structural, thermodynamic, and kinetic properties of the parallel G-quadruplexes formed by oligodeoxynucleotides d(G4T), d(TG4T), and d(TG5T), in which all phosphates were replaced with N-methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups, resulting in either negatively charged or neutral DNA sequences, respectively. The study showed that nucleic acids modified with N-methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups are compatible with G-quadruplex formation and established that all modified sequences form parallel G-quadruplexes. Compared to negatively charged G4s, the assembly of neutral G4 DNA species was faster in the presence of sodium ions than potassium ions but was independent of the salt concentration used. The formation of mixed G4s composed of both native and neutral G-rich strands was confirmed using native gel electrophoresis, size-exclusion chromatography, and ESI-MS.
Miroshnichenko et al. (2019) reported the synthesis and in vitro evaluation of mesyl phosphoramidate oligonucleotides. Mesyl phosphoramidate oligonucleotides recruit RNase H and exhibit significant advantages over phosphorothioate in RNA affinity, nuclease stability, and specificity in inhibiting key processes of carcinogenesis. The researchers reasoned that mesyl phosphoramidate oligonucleotides are an attractive alternative to phosphorothioates and reported the synthesis and in vitro evaluation of oligonucleotides in which the mesyl (methanesulfonyl) phosphoramidate group (µ-modification) was substituted for the natural PO group in each internucleotide position. The study compared the biological potency of miR-21 targeting ODNs spanning the full length of miR-21 (22 nt) and modified in each internucleotide position with that of either a µ- (µ-miR-21-ODN) or PS group (PS-miR-21-ODN). Unmodified ODN (PO-miR-21-ODN) served as a control. RNA duplexes with fully modified 2′-OMe or 2′-MOE RNAs, or LNAs, are not RNase H substrates; however, this study showed that a µ-ODN could elicit RNase H activity.
Anderson et al. (2021) evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages into the DNA gap and flanking regions of gapmer PS ASOs. The research group characterized the impact of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity, and toxicity in cells and in mice. The study showed that MsPA can replace all PS linkages in a gapmer ASO without compromising chemical stability or RNA-binding affinity, but these designs reduced activity.
Altman, S. & Angele-Martinez, C. (2021) investigated mesyl-modified oligonucleotides (MOs) for their ability to inactivate gene expression in bacteria. The researchers used an MO as a 5’-peptide conjugate targeting GyrA, an essential gene in bacteria that encodes DNA gyrase, a type II topoisomerase.
Hammond et al. (2021) evaluated a series of 2'-deoxy and novel 2'-O-methyl and 2'-O-(2-methoxyethyl) (2'-MOE) oligonucleotides with internucleotide methanesulfonyl (mesyl, μ) or 1-butanesulfonyl (busyl, β) phosphoramidate groups as potential splice-switching oligonucleotides. The study found no significant difference in splice-switching efficacy between 2'-MOE mesyl oligonucleotide and the corresponding phosphorothioate (nusinersen).
Gaponova et al. (2022) evaluated the antitumor potential of highly selective, multitarget antisense downregulation of small non-coding RNAs (microRNAs), which are frequently dysregulated in cells and can trigger oncotransformation and tumor development. The study found that the combination of recently developed mesyl phosphoramidate oligonucleotides, targeted to multifunctional miRNA regulators miR-17, miR-21, and miR-155, exhibited potent synergistic antiproliferative and antimigrative effects on highly aggressive tumor cells.
Proskurina et al. (2022) investigated the activating effects of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on ex vivo populations of immunocompetent cells. The μCpG ODNs used were modified with a palmitoyl group at the 5′-end at a maximum distance from the 3′-terminal palindrome. Unfortunately, μCpGs showed no antitumor activity when used for in situ vaccination.
Zhang et al. (2022) reported that a single 2′-O-methyl (2′-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2′-methoxyethyl, 2′ constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. Replacing the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without loss of potency and increased the elimination half-life of the ASOs. Zhang et al. found that two MsPA modifications at the 5′ end of the gap or in the 3′-wing of a Gap 2′-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap, without altering the safety profile, as observed across multiple sequences in cells and animals. Utilizing the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2′-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.
Pollak et al. (2023) observed that the placement of two mesyl phosphoramidate linkages within a PS ASO gap is the most promising strategy to mitigate PS ASO-dependent TLR9 activation, thereby enhancing the therapeutic index and further streamlining PS ASO drug development.
Sergeeva et al. (2024) identified patterns of mesyl or busyl modifications in ASOs for optimal knockdown in vitro using Malat1 lncRNA as a target. The combination of prostate-specific membrane antigen (PSMA) ligand-mediated delivery with optimized mesyl and busyl ASOs resulted in efficient target depletion in prostate cancer cells. This study showed that other N-alkanesulfonyl phosphoramidate groups can serve as essential components of mixed backbone gapmer ASOs, thereby mitigating the drawbacks of uniformly PS-modified gapmers.
More recently, Patutina et al. (2025) reviewed the use of modified synthetic nucleic acids, including MsPA, in catalytic nucleic acids, including miRNA-targeted ribozymes, DNAzymes/XNAzymes (antimiRzymes), and artificial ribonucleases (miRNases). Catalytic nucleic acids enable selective suppression of overexpressed miRNAs in pathological conditions through multiple enzymatic cleavage events.
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