RNA Modification Chemistry
PACE-Modified RNA
When maximum nuclease resistance matters, PACE end-modification delivers unmatched intracellular stability — without compromising the biological activity your experiments depend on.
Why PACE outperforms standard modifications
Performance Advantages
1–2 orders of magnitude higher intracellular stability vs. MS-modified counterparts over 24–96 hours (1,2)
2× or greater editing in Cas9 mRNA cotransfections in standard cell culture conditions (1)
Preserves and can increase gRNA specificity end modification maintains editing activity; internal modifications can reduce off-targets (4,5)
Up to 10× higher editing in human cells under nuclease-rich, serum-present conditions (1)
1.3× higher editing in RNP formats relative to MS-protected guides (3)
No liver toxicity or inflammatory response in vivo confirmed in mice after repeat dosings (4)
A backbone built for stability
The Chemistry Behind It
The PACE modification introduces a carbon–phosphorus bond into the oligonucleotide backbone — a linkage that is nearly impervious to nuclease cleavage while retaining a negatively charged backbone. This distinction is critical: the negative charge preserves the essential molecular recognition features that allow PACE-modified oligonucleotides to behave like their unmodified counterparts.
The result is native-like duplex formation, excellent aqueous solubility, and exceptional nuclease stability — achieved together, not at the expense of one another.
By contrast, 2′-O-methyl phosphorothioate (MS) chemistries provide more limited nuclease protection and are associated with increased nonspecific protein binding and off-target effects. PACE offers fundamentally superior backbone stability while maintaining native-like biophysical and biological behavior.
The PACE backbone at a glance
A non-bridging oxygen on the phosphate is replaced by a phosphonoacetate group (–CH₂–COOH), introducing a carbon–phosphorus (C–P) bond that is hydrolytically inert to nucleases.
Unlike phosphorothioate chemistries, PACE retains the native negative charge of the backbone, avoiding the nonspecific protein interactions that contribute to off-target toxicity.
Negatively charged · Nuclease-resistant · Native-like
| Property / Feature | 2′-O-Me PACE | 2′-O-Me PS | Phosphorothioate (PS) |
|---|---|---|---|
| Chemical type | Sugar + backbone2′-O-Me + phosphonoacetate | Sugar + backbone2′-O-Me + phosphorothioate | Backbone onlyphosphorothioate |
| Nuclease stability | Highest | Moderate to strong | Moderate |
| Duplex stability | Comparable to unmodified | Increases thermal stability | Comparable to unmodified |
| Nonspecific protein binding | Low | Moderate | Moderate to high |
| gRNA specificity | Maintained or improved4,5 | Maintained | Potential off-target effects |
| In vivo safety | No liver toxicity or inflammation observed4 | Variable | Dose-dependent toxicity concerns |
References
D.E. Ryan, et al., Biochemistry 2023, 62 (24), 3512–3520
D. Sheehan, et al., Nucleic Acids Res. 2003, 31, 4109–4118
Sousa, A.A., Hemez, C., Lei, L. et al. Nat. Biomed. Eng. 9, 7–21 (2025)
Rothgangl, T., Tálas, A., Ioannidi, E.I. et al. Nat. Biomed. Eng. 9, 1705–1718 (2025)
D.E. Ryan, et al., Nucleic Acids Res. 2018, 46, 792–803
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