The conjugation of C16 to siRNAs enables broad and efficient siRNA delivery to the CNS in rodents.
Brown et al. (2022) showed that conjugation of 2’-O-hexadecyl (C16) to siRNAs enables safe, potent, and durable silencing of genes in the central nervous system (CNS), the eye, and the lung in rodents and non-human primates, with a broad cell-type specificity. The goal of this study was to understand C16-siRNA uptake and RNAi activity across major CNS cell types. Brown et al. designed siRNAs against cell-type-specific targets uniquely expressed in neurons (Map2), astrocytes (Gfap), microglia (Iba1), or oligodendrocytes (Mbp) and the endothelium/perivascular macrophages (Pecam1).
The design of C16-siRNAs involves the conjugation of hexadecanoic acid or palmitic acid into the sense strand of the siRNA. The scientists introduced the selected C16 lipophilic moiety at the 2′-position of the ribose sugar on the sense strand, at position N6 from the 5'-terminal end. In this position, the C16 moiety maintains functional RISC activity while reducing the risk of exonuclease-mediated cleavage of the conjugate. Also, the strategic placement of 2′-fluoro and 2′-O-methyl chemical modifications or glycol nucleic acid (GNA) in the antisense seed region increased specificity and potency in the CNS. The addition of C16 and/or 5′-(E)-vinyl-phosphonate (VP), a 5′-phosphate mimic, placed at the 5′-end of the antisense strand, promoted RISC loading and potency in the spinal cord. An siRNA containing both VP and C16 showed the best activity across CNS regions, with a knockdown of mRNA of up to 90% and 75% in the spinal cord and brain, respectively.
The intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types. Sustained RNA interference (RNAi) activity lasted for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in potent, durable knockdown. This study also evaluated the preclinical efficacy of an siRNA targeting the amyloid precursor protein in a mouse model of Alzheimer’s disease. The siRNA treatment improved physiological and behavioral deficits.
Taken together, these results showed that C16 conjugation of siRNAs allows the design of safe therapeutic silencing drugs for targeting genes outside the liver.
Figure 1: Chemical structures of 2’-O-C16, 5’-€-VP, and GNAs.These modifications enhance extrahepatic delivery, particularly to the CNS, the heart, skeletal muscle, and lungs, by facilitating binding to human serum albumin (HSA).
Guidelines for designing effective C16-siRNA conjugates
[1] Link C16 to the nucleic acid at position six of the sense strand
The placement of the C16 lipid is the most critical design choice. The conjugation site selection is critical. Often, the C16 moiety is attached to the 5' end of the sense (passenger) strand. However, as observed by Brown et al. (2026) for the delivery to the CNS, the C16 moiety is linked to the nucleic acid at position six of the sense strand. Attaching it to the antisense strand usually disrupts RISC loading and gene silencing.
[2] If placed at the 5’-end
For placement at the 5’-end, select a rigid or flexible linker, such as a prolinol or PEG linker, between the C16 chain and the oligonucleotide to prevent the lipid from sterically hindering the RNA's ability to duplex or interact with proteins. Lipid-conjugated siRNAs stay in systemic circulation longer, where they are exposed to exonucleases for extended periods; hence, a mixed modification pattern is preferred.
[3] Add PS or phosphorothioate linkages: Incorporate PS linkages at the terminal ends, usually at the first 2 to 3 bases of both 5'- and 3'-ends, to protect against exonuclease degradation.
[4] Select Sugar Modifications: Use a mixed combination of 2'-F (2'-fluoro) and 2'-OMe (2'-O-methyl) modifications across the entire length of both strands. Follow an alternating pattern or a "zipper" motif to maximize thermal stability (Tm) and metabolic resistance.
[5] Sequence Selection Criteria: The siRNA sequence must be optimized for targeting potency before adding the C16 modification.
- Typically, a siRNA has 19 to 21 base pairs with 2-nucleotide 3'-overhangs.
- Aim for 30% to 50% GC content. High GC content can lead to toxicities or poor RISC loading.
- Ensure the 5'-end of the antisense strand is less stable (lower binding energy) than the 5'-end of the sense strand to ensure correct RISC loading.
- Check for off target matches in the 3' UTR of non-target genes, especially for the seed region around nucleotides 2 to 8.
Physicochemical Considerations
- The addition of C16 significantly increases the molecule's hydrophobicity. During purification, for example, using HPLC, a C16-siRNA will elute much later than a standard siRNA.
- The design relies on the C16 chain binding noncovalently to albumin. Ensure that buffer conditions during in vitro testing include serum, or the results won't accurately reflect in vivo performance.
As reported by Brown et al. (2022) and Titze-de-Almeida et al. (2026), the incorporation of a 16-carbon chain into siRNA (C16-siRNA) enhanced its delivery to the central nervous system (CNS), as a result of enhanced penetration and distribution within the CNS by modulating lipid–protein interactions, endocytic uptake, and parenchymal diffusion.
Furthermore, C16-siRNAs also demonstrated notable biodistribution in ocular and pulmonary tissues. The addition of a C16 group to siRNAs expands the potential for the design of therapeutic siRNAs that address diseases affecting multiple important organs.
In designing an amyloid precursor protein (APP)-targeting siRNA conjugate, Brown et al. considered improvements in specificity and duration of action based on previously FDA-approved drugs.
The siRNA-conjugate with the best activity across CNS regions combined VP and C16, achieving up to 90% and 75% mRNA knockdown in the spinal cord and brain, respectively.
Design example siRNA-conjugate XVIII targeting APP (Brown et al. 2022)
Sense 5’-u•a•uga(C16a)GuUCAucaucaaa•a•a-3’
Antisense 5’-VP-u•U•uuugAugaugaAcUucaua•u•c-3’
• = PS, (C16n) = 2′-O-C16, A = GNA, lower case = 2′-O-methyl (2′-OMe) ribosugar modifications,, upper-case = 2′-deoxy-2′-fluoro (2′-F), VP = 5'-(E)-vinylphosphonate.
Design example of a C16-siRNA for CNS delivery
Legend: * = PS, N6-C16 = NA-2′-O-C16, N = GNA, n lower case = 2′-O-methyl (2′-OMe) ribosugar modifications, N upper-case = 2′-deoxy-2′-fluoro (2′-F), VP = 5'-(E)-vinyl-phosphonate.
Figure 2. Structure of Human APP-Targeting siRNA XVIII. This illustration depicts the C16-siRNA structure, as discribed in Brown et al. (2022
Key components include:
(i) the sixteen carbon atoms of the lipophilic moiety (C16),
(ii) the conjugation of the C16 ligand to the 2’ position of the pentose in the adenine nucleotide (blue color),
(iii) upper-case and lower-case letters denoting the 2′-deoxy-2′-fluoro (2′-F) and 2′-O-methyl (2′-OMe) ribosugar modifications, respectively;
(iv) vertical blue lines connecting the sense and antisense strands, illustrating Watson and Crick hydrogen bonds between complementary nucleotides,
(v) uppercase letter N (red) in the antisense strand indicates a modification of glycol nucleic acid (GNA),
(vi) the symbol (•) indicates phosphorothioate (PS) linkages, and
(vii) “VP,” refers to the incorporation of 5’-(E)-vinylphosphonate.
Abbreviations: C16, 16-carbon-length molecule; VP, vinylphosphonate.
References
Alnylam Pharmaceutics, Inc. Alnylam. Delivery Platforms—C16 Conjugates. Available online: sirna-delivery-platforms.
Brown, K.M.; Nair, J.K.; Janas, M.M.; Anglero-Rodriguez, Y.I.; Dang, L.T.H.; Peng, H.; Theile, C.S.; Castellanos-Rizaldos, E.; Brown, C.; Foster, D.; et al. Expanding RNAi Therapeutics to Extrahepatic Tissues with Lipophilic Conjugates. Nat. Biotechnol. 2022, 40, 1500–1508. [PubMed]
Titze-de-Almeida, R., Oliveira Gomes, G. d. M., Santos, T. C. d., & Titze-de-Almeida, S. S. (2026). C16-siRNAs in Focus: Development of ALN-APP, a Promising RNAi-Based Therapeutic for Alzheimer’s Disease. Pharmaceuticals, 19(1), 26. C16-siRNAs in Focus
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