Boost Protein Expression With Poly(A) Innovation

trilinkbiotech.com | 1 Improved total protein expression and duration with ModTail™ technology, a novel poly(A) tail modification Introduction Recent innovations in mRNA design have accelerated the clinical application of several mRNA-based therapeutics. TriLink’s CleanCap® technology facilitated the development of BNT162b1, the highly successful Pfizer/BioNTech COVID-19 vaccine. The cap and the use of modified nucleotides were key to the success of mRNA vaccines. However, it is widely recognized that further innovations are required to optimize mRNA molecules to meet the requirements for wider therapeutic applications. CleanCap® M6 technology, TriLink’s most robust cap analog to date, has demonstrated substantially increased protein expression in vivo compared with mRNAs using existing industry standards. The increase in protein expression is, at least in part, due to decreased Dcp2-mediated decapping.1 CleanCap M6 may help increase the potency of mRNA-based medicines with the benefit of acheiving higher expression at the same dose. At the 3′ end of mRNA, researchers are exploring modifications to the poly(A) tail to enhance resistance against exonuclease activity.2,3,4 This approach aims to prolong the duration and increase overall expression of proteins from mRNA therapies. To pursue this strategy, we synthesized and tested numerous potential poly(A) tail modifications. After thorough evaluations, the most promising candidate was selected. This technical note introduces the ModTail™ technology, TriLink's novel poly(A) tail modification, which is shown to increase total protein expression and extend the duration of expression of various N1-methylpseudouridine (N1MePsU)-mRNA constructs in both in vitro cell assays and in vivo studies using mice. Results and discussion Using the screening protocols as illustrated in Figure 1 for mRNA tail modification, starting with a purified EGFP mRNA, a variety of tail modifications were added to the poly(A) tail of the EGFP mRNA and were screened for mRNA duration and total protein expression. From those screening experiments, our results show the ModTail poly(A) tail modification significantly increases total protein expression and expression duration. In vivo mouse studies were also performed to confirm the impact of tail modification on mRNA translation and longevity. Figure 1. Tail modification screening process flow. In vitro-transcribed mRNA was purified, and then the poly(A) tail modification was added. Tail-modified mRNA is purified again before in vitro cell assays and in vivo mouse studies. AAAAA Addition of poly(A) tail modification Purification 5′UTR Open reading frame 3′UTR 5′UTR Open reading frame 3′UTR In vitro cell assay In vivo studies AAAAA Technical note trilinkbiotech.com | 2 Table 1 shows a side-by-side mRNA quality comparison of N1-methylpseudouridine (N1MePsU)-EGFP mRNAs with or without the ModTail technology. Both mRNAs have comparable capping efficiency, purity, and dsRNA level. The tail modification was incorporated with high efficiency. Table 1. Measurements of capping efficiency, purity, dsRNA quantity, and incorporation efficiency in CleanCap M6 EGFP mRNA with and without the ModTail technology from Figure 2. N1MePsUmodified construct Capping efficiency (%)* Purity (%)** dsRNA quantity (ng per µg RNA)*** ModTail technology incorporation efficiency (%)** CleanCap M6 EGFP mRNA 96.6 98.5 0.41 N/A CleanCap M6 EGFP ModTail mRNA 96.5 99.7 0.44 99.7 *Measured by LC-MS after ribozyme digestion **Measured by IP-RP HPLC analysis ***dsRNA was measured by ELISA Next, the two EGFP mRNAs were transfected into 293T cells, and the resulting fluorescence was monitored over time for 96 hours. Figure 2 shows a distinct increase in total EGFP protein expression for ModTail mRNA over unmodified mRNA. Also, peak protein expression is shifted to a longer time point for the EGFP ModTail mRNA. Figure 2. Introduction of ModTail™ technology to CleanCap® M6 EGFP mRNA increases total protein expression in 293T cells. In blue, fluorescence readout of EGFP expression in 293T cells from CleanCap M6 EGFP mRNA. In orange, fluorescence readout of EGFP expression in 293T cells from CleanCap M6 EGFP mRNA with ModTail technology. Fluorescence intensity is measured as total integrated green object intensity (GCU x μm2/image). The values are shown as mean ± standard deviation, p =0.0014 by Student’s t-test from triplicate samples. 0 8 16 24 32 40 48 56 64 72 80 88 96 0 2x106 4x106 6x106 8x106 1x107 293T cells Time (hrs) Fluorescence intensity EGFP ModTail mRNA EGFP mRNA trilinkbiotech.com | 3 0 8 16 24 32 40 48 56 64 72 80 88 96 0 1x106 2x106 3x106 4x106 5x106 Time (hrs) A 40A B C 100A D 120A 60A Fluorescence intensity Fluorescence intensity Fluorescence intensity Fluorescence intensity EGFP mRNA EGFP ModTail mRNA 0 8 16 24 32 40 48 56 64 72 80 88 96 0 1x106 2x106 3x106 4x106 5x106 Time (hrs) 0 8 16 24 32 40 48 56 64 72 80 88 96 0 1x106 2x106 3x106 4x106 5x106 Time (hrs) 0 8 16 24 32 40 48 56 64 72 80 88 96 0 1x106 2x106 3x106 4x106 5x106 Time (hrs) Next, we explored whether ModTail™ technology is compatible with different poly(A) tail lengths. CleanCap® M6 EGFP mRNAs with tail lengths of 40A, 60A, 100A, and 120A, and with or without the ModTail technology, were transfected into 293T cells. The resulting fluorescence was monitored over 72 hours. For each tail length, the ModTail mRNA increased protein expression and increased the duration of expression (Figure 3). This demonstrates the compatibility of ModTail mRNA with a wide range of poly(A) tail lengths. Figure 3. ModTail mRNA with different poly(A) tail lengths provides similar increases in duration and total protein expression in 293T cells. Fluorescence readout of EGFP expression in 293T cells from CleanCap M6 EGFP mRNA with poly(A) tail lengths of (A) 40A, (B) 60A, (C) 100A, and (D) 120A, either with (orange) or without (blue) the ModTail technology. The fluorescence intensity values are shown as mean ± standard deviation, p<0.0001 by two-way ANOVA followed by Tukey's multiple comparison test in triplicate samples. Figure 4. The introduction of ModTail technology to EGFP mRNA with different caps results in similar increases in total protein expression in 293T cells. (A) Fluorescence readout of EGFP expression in 293T cells of 5 ng CleanCap AG 3′ OMe EGFP mRNA with (orange) and without (blue) ModTail technology. (B) Fluorescence readout of EGFP expression in 293T cells of 5 ng CleanCap M6 EGFP mRNA with (orange) and without (blue) the ModTail technology. 0 8 16 24 32 40 48 56 64 72 80 88 96 0 2x106 4x106 6x106 8x106 1x107 Time (hrs) CleanCap AG 3′ OMe EGFP mRNA CleanCap AG 3′ OMe EGFP ModTail mRNA 0 8 16 24 32 40 48 56 64 72 80 88 96 0 2x106 4x106 6x106 8x106 1x107 293T cells Time (hrs) Fluorescence intensity CleanCap M6 EGFP ModTail mRNA CleanCap M6 EGFP mRNA Fluorescence intensity A 293T cells B trilinkbiotech.com | 4 So far, we have confirmed the ModTail™ technology is compatible with different mRNA constructs and poly(A) tail lengths. We also wanted to determine if ModTail technology is compatible with different mRNA caps. EGFP mRNAs with CleanCap® M6 or CleanCap AG 3′ OMe with or without the ModTail technology were transfected into 293T cells. The resulting fluorescence signal was monitored over time for 96 hours. Figure 4 shows that both mRNAs with the ModTail technology showed increased protein expression and a shift of peak protein expression to a later time point. This ModTail technology was also tested in various other cell types, including A549 and activated PBMCs. Similar results were obtained for each cell type. Figure 5. Mice injected with FLuc ModTail mRNA show increased duration of protein expression. (A) Representative images of FLuc levels at 12, 48, and 72 hr in mice injected with 0.3 milligrams per kilogram (mpk) of LNP formulated CleanCap M6 mRNA with or without the ModTail technology (B). Representative images of FLuc levels at 12, 48, and 72 hr in mice injected with 0.3 mpk of LNP formulated CleanCap AG 3′ OMe mRNA with or without ModTail modification. (C) Time course of FLuc levels in mice injected with 0.3 mpk of LNP formulated CleanCap M6 mRNA with or without ModTail. Results shown are from repeated measures ANOVA (two-tailed), P=0.0013. Tukey’s multiple comparisons test at 12 hrs, P=0.3692, and 48 hrs, P=0.0009, and 72 hrs, P=0.3313. Values for luminescent intensity (photon/s) are shown as mean ± standard error (n=7 animals per group). (D) Time course of FLuc levels in mice injected with 0.3 mpk of LNP formulated CleanCap AG 3′ OMe mRNA with or without ModTail technology. Results of repeated measures ANOVA (two-tailed), P=0.0209. Tukey’s multiple comparisons test at 12 hrs, P=0.0974, and at 48 hrs, P=0.0488, and at 72 hrs, P=0.6344. Values for luminescent intensity (photon/s) are shown as mean ± standard error (n=7 animals per group). 036 12 24 48 72 96 120 144 168 2.7x108 1.1x109 4.3x109 1.7x109 6.9x1010 2.8x1011 1.1x1012 CleanCap M6 mRNA CleanCap M6 ModTail mRNA Luminescent intensity (photon/s) PBS CleanCap M6 mRNA CleanCap M6 ModTail mRNA PBS CleanCap AG 3' OMe mRNA CleanCap AG 3' OMe ModTail mRNA Luminescent intensity (photon/s) Time (hrs) Time (hrs) 12 48 72 hrs 12 48 72 hrs CleanCap AG 3' OMe ModTail mRNA CleanCap AG 3' OMe mRNA 036 12 24 48 72 96 120 144 168 1.7x107 6.7x107 2.7x108 1.1x109 4.3x109 1.7x1010 6.9x1010 2.7x1011 1.1x1012 C A B D trilinkbiotech.com | 5 Next, we checked to see if the effects of the ModTail™ technology seen in vitro would translate to in vivo using a mouse model. LNP formulated CleanCap® M6 or CleanCap AG 3′ OMe FLuc mRNAs with or without the ModTail technology were injected into the tail vein of mice. The resulting FLuc levels were monitored over time. Images of mice injected with PBS, FLuc mRNA, or FLuc ModTail mRNA are shown at 12, 48, and 72 hours, demonstrating the increased duration of protein expression in vivo (Figure 5). Table 2. Measurements of capping efficiency, purity, dsRNA quantity, and incorporation efficiency in FLuc mRNA with and without the ModTail technology from constructs shown in Figure 5. N1MePsU-modified FLuc construct Capping efficiency (%)* Purity (%)** dsRNA quantity (ng per µg RNA)*** ModTail technology incorporation efficiency (%)** CleanCap AG 3′ OMe mRNA 98.0 97.5 0.6 N/A CleanCap AG 3′ OMe ModTail mRNA 98.0 95.4 0.6 98.9 CleanCap M6 mRNA 99.0 98.8 0.4 N/A CleanCap M6 ModTail mRNA 98.8 97.2 0.5 99.5 *Measured by LC-MS after ribozyme digestion **Measured by IP-RP HPLC analysis ***dsRNA was measured by ELISA Figure 6. Mice injected with hEPO ModTail mRNA show increased duration of protein expression. C57BL/6 mice were administered via tail vein injection with 0.1 mpk LNP formulated CleanCap M6 mRNAs encoding hEPO with (orange) or without (blue) the ModTail technology. (A) Serum hEPO levels were measured by ELISA and plotted by time point. (B) The area under the curve was calculated by integrating all time points from 5 separate animals per group. Both mRNAs have comparable capping efficiency (99.5%), purity, and dsRNA level (1 ng/µg mRNA). 1 4 16 64 256 1024 4096 16384 Time post-injection (hrs) hEPO mRNA hEPO ModTail mRNA 0 6 12 24 48 72 96 AUC0-96 hrs Serum hEPO expression Serum hEPO (µg/mL) A B 0 1x105 2x105 3x105 4x105 5x105 6x105 hEPO ModTail mRNA hEPO mRNA ~1.25-fold trilinkbiotech.com | 6 Figure 7. ModTail™ mRNA does not increase inflammatory immune responses in vivo. Serum levels of (A) IFNα, (B) IL-6, (C) IP-10, and (D) MCP-1 levels at 1, 6, and 24 hrs post-dosing for mice administered hEPO mRNA with or without ModTail technology as described in Figure 6. 0.1 mpk mRNA-LNP capped with ARCA without any modification measured as reference and is shown in red dashed line. ARCA was chosen as a reference because its use results in Cap 0 mRNA, which is immunogenic.5 Whiskers show min to max, and the line shows the median. Dots show five separate animals and their experimental duplicates per group. Besides the reporter gene FLuc mRNA, we also conducted an in vivo mouse study with human erythropoietin (hEPO) mRNA (Figure 6). Again, we confirmed that the tail modification enhanced the overall magnitude and duration of hEPO protein expression in vivo. Also, we evaluated whether the tail modification triggered any extra innate immune response. We checked the expression levels of two inflammatory cytokines (IFN-α and IL-6) and two chemokines (IP-10 and MCP-1) in mouse serum at different time points: 1, 6, and 24 hrs after injection (Figure 7). No significant differences were observed between mRNAs with or without tail modification, and biomarkers remained well below levels that would indicate an inflammatory immune response. IP-10 MCP-1 IL-6 (pg/mL) IP-10 (pg/mL) MCP-1 (pg/mL) IFN-α (pg/mL) 1h 6h 24h 0 100 200 300 400 500 10000 20000 30000 1h 6h 24h 0 100 200 300 400 2000 4000 6000 IFN-α IL-6 WT U + ARCA WT U + ARCA WT U + ARCA WT U + ARCA 1h 6h 24h 0 200 400 600 800 5000 10000 15000 PBS hEPO mRNA hEPO ModTail mRNA 1h 6h 24h 0 100 200 300 400 5000 10000 10770 Wateridge Circle, Suite 200 San Diego, CA 92121 | 800.863.6801 | trilinkbiotech.com © 2025 TriLink BioTechnologies. All rights reserved. TriLink, CleanCap, is a trademark of TriLink BioTechnologies. 091625 Conclusion In this technical note, we demonstrate the ease of use, efficacy, and flexibility of ModTail™ technology. ModTail technology is compatible with various tail lengths, cap structures, and mRNA constructs. Additionally, it is easy to detect the ModTail technology incorporation efficiency via IP-RP HPLC analysis. ModTail technology increased total protein expression and the duration of expression in both in vitro cell assays and in vivo mouse studies with different mRNA constructs. The resulting higher protein expression and longer expression duration can potentially improve mRNA therapeutics. Higher protein expression potentially means lower dosage of mRNA for the same effect. Also, longer duration can lead to less frequent mRNA dosing for therapeutic use, which could translate to reduced inflammatory responses and fewer side effects. Together with cap modification, base modification, and mRNA sequence optimization, ModTail technology can help enable the next-generation mRNA medicine. Acknowledgements Allele Biotechnology performed the cell assay work for the different tail lengths experiment (Figure 3). References 1. Mandell ZF and Ujita A et al. (2025) CleanCap® M6 inhibits decapping of exogenously delivered IVT mRNA. Molecular Therapy Nucleic Acids (1), 102456. https://pubmed.ncbi.nlm.nih.gov/39974289/ 2. Aditham A et al. (2022) Chemically modified mocRNAs for highly efficient protein expression in mammalian cells. ACS Chemical Biology, 17, 3352-3366. https://pubmed.ncbi.nlm.nih.gov/34995053/ 3. Chen H et al. (2025) Branched chemically modified poly(A) tails enhance the translation capacity of mRNA. Nature Biotechnology, 43, 194-205. https://pubmed.ncbi.nlm.nih.gov/38519719/ 4. Li CY et al. (2022) Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cells and in vivo. Mol Ther Nucleic Acids 30: 300–310. https://pubmed.ncbi.nlm.nih.gov/36320322/ 5. Zhong Z et al. (2018) mRNA therapeutics deliver a hopeful message. Nano Today 23: 16–39. https://doi.org/10.1016/j. nantod.2018.10.005