NADES as Biocompatible Media for Thermally Stable RNA Molecules

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results NADES as Biocompatible Media for Thermally Stable RNA Molecules View ORCID Profile Lamya Al Fuhaid , View ORCID Profile Shahryar Khattak , View ORCID Profile Arwa Alghuneim , View ORCID Profile Imed Gallouzi , View ORCID Profile Young Hae Choi , View ORCID Profile Robert Verpoorte , View ORCID Profile Geert-Jan Witkamp , View ORCID Profile Andreia Farinha doi: https://doi.org/10.1101/2025.07.23.665770 Lamya Al Fuhaid a Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology , Thuwal, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lamya Al Fuhaid For correspondence: lamya.fuhaid{at}kaust.edu.sa Shahryar Khattak a Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology , Thuwal, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shahryar Khattak Arwa Alghuneim a Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology , Thuwal, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arwa Alghuneim Imed Gallouzi a Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology , Thuwal, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Imed Gallouzi Young Hae Choi b Institute Biology Leiden, Leiden University , Leiden, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Young Hae Choi Robert Verpoorte b Institute Biology Leiden, Leiden University , Leiden, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert Verpoorte Geert-Jan Witkamp a Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology , Thuwal, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Geert-Jan Witkamp Andreia Farinha a Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology , Thuwal, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andreia Farinha Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The inherently low thermal stability of RNA poses operational, accessibility, and financial challenges for RNA research and RNA-based technologies. Natural deep eutectic solvents (NADES) have been shown to enhance the stability of various macromolecules, including DNA and protein. This study explores NADES as alternative storage media for RNA preservation and evaluates their biocompatibility in human cells. In vitro -transcribed mRNA was stored in various NADES at temperatures ranging from 4−50 °C, and the integrity was assessed by quantifying its translatability, represented by protein-expressing cells. NADES effectively preserved RNA integrity, retaining over 50% of its translatability for at least four months at room temperature (21 °C) and 48 hours at 50 °C, in comparison to −80 °C storage. Notably, the preservation efficacy remained unaffected by temperature fluctuations. Furthermore, the selected NADES concentrations maintained ∼ 99% cell viability, demonstrating their biocompatibility. These findings establish NADES as efficient, biocompatible RNA storage media, enabling stable storage and transport at ambient and extreme temperatures while withstanding sudden fluctuations. This enhanced stability simplifies and expands the accessibility of RNA-based applications. Additionally, the biocompatibility of NADES supports their potential use in RNA-based biomedical applications. 1. Introduction The stability of RNA is critical for RNA-related research and downstream applications. Despite a few variations, DNA and RNA are composed of similar building blocks and have similar structures. As opposed to the double-stranded structure of DNA, RNA is often single-stranded. The inter-strand hydrogen bonding in DNA can lower the total energy and protect the side chains from reactions 1 . Additionally, RNA contains ribose sugar that has an additional hydroxyl group compared to the deoxyribose sugar in DNA. The hydroxyl group can become deprotonated in basic pH conditions, generating an oxygen nucleophile 2 . The oxygen nucleophile may attack the adjacent phosphorus, breaking the phosphodiester bond in the RNA backbone, resulting in RNA hydrolysis 2 . Because of their low stability, RNA molecules are conventionally stored at low temperatures (approximately −80 °C) to maintain their integrity. Nowadays, messenger RNA (mRNA) vaccines are being developed for a myriad of infectious diseases. The efficacy of mRNA vaccines on mitigating diseases has been recently demonstrated during the COVID-19 pandemic. The potential of mRNA vaccines goes far beyond infectious diseases as they are currently employed for cancer immunotherapy 3 . They are also being developed for other deadly diseases, including human immunodeficiency virus (HIV) and malaria 4 , 5 . Despite the robustness of mRNA vaccines, the fragility of RNA molecules requires special freezing units of extremely cold temperatures (−90 to −60 °C). For example, according to the European Medicine Agency (EMA), two key COVID-19 mRNA vaccines, Moderna and BioNTech, are stable for 6 months in frozen conditions, but only up to 6 h at room temperature (RT) 6 .The need to store vaccines in frozen conditions complicates their transport and administration processes. Additionally, the ultra-cold storage units are expensive, complicated to use, and unequally accessible. As a result, a large percentage of mRNA vaccines lose their functionality during transportation or due to improper storage. These barriers create an imparity in the distribution of vaccines, putting developing countries at risk of not receiving proper health care. Several methods have been developed to improve the stability of pharmaceutical RNA formulations. For example, it has been shown that the 5’ linkages of uridine residues in the mRNA molecule are particularly susceptible to degradation 7 . Thus, uridine residues in in vitro -transcribed mRNA molecules have been substituted with pseudouridine or N 1 -methyl-pseudouridine 7 . Alternatively, uridine can be depleted through guanine/cytosine (G/C) content increase, which can consequently increase the thermodynamic stability of the mRNA 7 . Nevertheless, some chemical modifications can alter the biological properties of the molecule, which may introduce immunogenicity. Additionally, most studies investigating chemically modified mRNA focus on examining their cellular and in vivo performances without evaluating their storage stability or shelf life. Moreover, despite the implementation of pseudouridine substitution in some COVID-19 mRNA vaccines, their stability at RT is still limited. Deep eutectic solvents (DES; singular and plural) are green solvents formulated from non-toxic, low-cost materials 8 , 9 . They are composed of hydrogen-bonded components, forming low-melting-point liquids with low moisture content 10 . DES are thermally stable, non-volatile, biodegradable, and easy to prepare 8 , 9 . They have unique supramolecular structures and properties, linking them to diverse applications. For example, DES have been used in the fields of catalysis 11 , extraction 12 – 15 , and dissolution of pharmaceutical ingredients 16 . DES made from natural metabolites, like sugars, amino acids, and organic acids, are subtyped as natural DES (NADES) 17 . The components of NADES are mostly accessible in food-grade quality. NADES are often biocompatible and have a lower environmental impact than other DES, presenting a superior option for biomedical and environmental applications 18 . Due to their wide polarity range, DES can dissolve and stabilize various compounds, such as rutin 13 , paclitaxel (Taxol) 14 , and macromolecules, including DNA 19 – 21 , protein 15 , 22 , 23 , and starch 24 . The stabilization capacity of DES is partially attributed to their hydrogen bond network, which lowers the free energy and prevents macromolecule aggregation. The low water activity in DES was also linked to macromolecule stability 25 . An example of macromolecule stabilization was demonstrated by the capacity of choline chloride-glycerol and choline chloride-ethylene glycol DES to maintain DNA for six months at RT 21 . It was proposed that the cations in choline chloride could have participated in the solvation process by interacting with the negatively charged phosphate groups 10 , 21 . In this work, NADES are proposed as an alternative medium for storing RNA at RT. The stability of in vitro -transcribed model RNA stored in NADES at temperatures ranging from 4−50 °C is examined. Additionally, the translatability of the mRNA in cellulo at multiple time points after being stored in NADES is evaluated. The proposed solution does not include additional chemical modifications to the RNA or active alteration of its secondary structure. 2. Methods 1.2. NADES preparation and characterization NADES combining betaine (NADES 1−5) and choline chloride (NADES A−E) with glycerol and/or disaccharides were prepared using the heating method reported by Y. Dai et al ., 2013 14 . The stability of the resulting samples was evaluated over time and only those that did not exhibit crystallization within a week of formation were selected. All NADES were autoclaved prior to their use in tissue culture experiments. 2.2. Cell culture and viability test Human HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/L D-glucose and GlutaMAX, GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma), 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO), according to standard protocols. Maximum working concentrations of NADES were determined based on a viability test in HeLa cells. Cells were seeded in 24-well plates at a density of 3.0×10 4 cell/well. When the cells reached ∼ 70% confluence, they were treated with varying concentrations of NADES. Cell viability was assessed 24 h post-treatment using the Invitrogen™ EVOS™ Digital Color Fluorescence Microscope and compared to untreated controls. Cytotoxicity at the selected concentration was further evaluated by quantifying the percentage of viable cells via flow cytometry (Becton Dickinson LSRFortessa™) 24 h post-treatment. 2.3. RNA in vitro transcription A plasmid encoding the mCherry open reading frame (ORF), including a T7 promoter, poly-A tail, and T7 terminator (Figure S1), was linearized with SacI-HF (New England Biolabs) overnight and purified using a PCR purification kit (QIAGEN). The linearized plasmid served as a template for in vitro transcription using the HighYield T7 ARCA mRNA Synthesis Kit (m5CTP/Ψ-UTP; Jena Biosciences), following the manufacturer’s instructions, to generate 5-methylcytidine and pseudouridine-modified mCherry mRNA. The transcribed RNA was purified using the Monarch RNA Cleanup Kit (New England Biolabs), aliquoted on ice, and stored at −80°C. The undigested plasmid, linearized plasmid, in vitro –transcribed mRNA, and polyadenylated mRNA were visualized on a 1.2% agarose gel (Figure S2). 2.4. mRNA transfection HeLa cells were seeded in six-well plates at a density of 1.2×10 5 cells/well and transfected 48 h later with 250 ng mCherry mRNA using Lipofectamine MessengerMAX™ Transfection Reagent (Thermo Fisher Scientific), following the manufacturer’s instructions. Cells were harvested and fixed 18 h post-transfection. The percentage of mCherry-expressing cells was quantified via flow cytometry (Becton Dickinson LSRFortessa™) using a Texas Red filter.. 3. Results and discussion 3.1. NADES exhibit low cytotoxicity in human cell culture models HeLa cells were cultured and treated with ten different concentrations of NADES, ranging from 2–20 µg/mL, and observed after 24 h. The maximum concentrations that did not result in a visible reduction in the cell viability were selected. The percentage of viable cells was then quantified by flow cytometry and was above 98% ( Figure 1 ). These results confirm the general biocompatibility of NADES with human cell culture models, in line with previous reports 18 . They also demonstrate the specific compatibility of the tested formulations and concentrations. Collectively, the findings suggest that NADES may be safe for use in biomedical applications. Download figure Open in new tab Figure 1. Cell viability HeLa cells treated with NADES Values are presented as mean ± SD for n=3. 3.2. NADES does not interfere with the in cellulo translation of mRNA To assess the ability of NADES to enhance the stability of mRNA without interfering with its translatability, the translation of mRNA into functional proteins after storage in NADES was evaluated. In vitro -transcribed mCherry mRNA was used as a model, as it encodes a red fluorescent protein, offering an easy detection and quantification through fluorescence measurement. mCherry is a widely used monomeric fluorophore model protein that absorbs light between 540–590 nm and emits in the 550–650 nm range. The in vitro -transcribed mCherry mRNA was stored in water and NADES at 37 °C, a biologically relevant temperature. The in cellulo translatability of the mRNA was evaluated after 48 h of storage at 37 °C. HeLa cells were transfected with the mRNA and the percentage of cells expressing mCherry protein was quantified by flow cytometry. Results were normalized to cells transfected with control mRNA stored at −80 °C. NADES based on betaine (NADES 1−5) were ineffective as mRNA storage media, interfering with the mRNA integrity and/or translatability ( Figure 2 ). Conversely, most of the tested choline chloride-based NADES (A, C, D, and E) supported efficient translation. NADES B was extremely viscous, which complicated handling and may have contributed to the low translation level observed after storage. These results indicate that different NADES formulations have varying effects on mRNA stability, with choline chloride-based systems demonstrating superior retention of integrity and functionality. Because NADES C showed the highest translation level, it was selected for the following experiments. Download figure Open in new tab Figure 2. mRNA translation after 48 h incubation at 37°C mCherry mRNA was incubated in water and NADES 1−5 and A−E, then transfected in HeLa cells. The percentage of cells expressing mCherry protein was quantified by flow cytometry and normalized to the percentage from cells transfected with control mCherry mRNA stored at −80 °C. 3.3. NADES protects RNA from degradation at elevated temperatures The integrity and in cellulo translatability of the mRNA was evaluated after 24 and 48 h of storage at 37 °C. HeLa cells were transfected with the mRNA, and the percentage of mCherry-positive cells was normalized to the percentage of positive cells transfected with control mRNA stored at −80 °C. Compared to the control, there was a slight drop in the percentage of positive cells by ∼ 10 and 15% for mRNA stored in NADES and water, respectively after storage for 24 h at 37 °C ( Figure 3 ). An additional 24 h of storage at the same temperature did not cause further reduction. Notably, the used mRNA includes a modified bases, resembling the mRNA molecules currently used in vaccines. This modification infers added stability to the mRNA, reducing its degradation at slightly elevated temperatures. Download figure Open in new tab Figure 3. mRNA translation after incubation at 37°C mCherry mRNA was stored in water and NADES at 37 °C then transfected into HeLa cells. The percentage of fluorescent cells was quantified and normalized to transfection with control mRNA stored at −80 °C. Values are presented as means ± SD for n=3, and the dashed lines represent non-linear one-phase decay fit curves. Moreover, to test the ability of NADES to protect RNA under extreme conditions, mRNA was also stored at 50°C. This temperature was selected to simulate harsh environments, such as those encountered during vaccine transport, to test the efficiency of NADES as a vaccine transportation medium. When stored in water, mRNA lost more than half its functionality after 24 h and dropped to ∼ 15% after 48 h compared to control ( Figure 4 ). In contrast, mRNA stored in NADES retained ∼ 70 and 50% of its functionality after 24 and 48 h storage at 50 °C, respectively. Collectively, these results highlight the superior protective effect of NADES over conventional storage media and demonstrate their remarkable ability to preserve RNA integrity even under extremely harsh environmental conditions. Download figure Open in new tab Figure 4. mRNA translation after incubation at 50°C mCherry mRNA was stored in water and NADES at 50 °C then transfected into HeLa cells. The percentage of fluorescent cells was quantified and normalized to transfection with control mRNA stored at −80 °C. Values are presented as means ± SD for n=3, and the dashed lines represent non-linear one-phase decay fit curves. 3.4. Temperature fluctuations do not affect RNA stored in NADES During a realistic transportation scenario, temperature fluctuations can occur abruptly. To examine the ability of NADES to protect RNA from such changes, mRNA was subjected to four temperature shifts: 12 h at 50 °C, 12 h at 4 °C, followed by another 12 h at 50 °C, and a final 12 h at 4 °C. When RNA was stored in NADES, there was a slight drop in translation after the first 50 °C exposure, but levels remained largely stable afterward ( Figure 5 ). In contrast, RNA stored in water lost almost all functionality after the initial 50 °C exposure. These results clearly demonstrate that drastic temperature fluctuations do not compromise the integrity of mRNA stored in NADES. This underscores the strong potential of NADES for effectively protecting RNA-based formulations and vaccines during transportation, even under harsh and unpredictable conditions. Download figure Open in new tab Figure 5. mRNA translation after fluctuating temperature storage mCherry mRNA was stored at fluctuating temperature for a total of 48 h and evaluated at 12-h intervals. The mRNA was transfected into HeLa cells, and the percentage of fluorescent cells was quantified. Data are normalized to transfection with control mRNA stored at −80 °C. Values are presented as means ± SD for n=3, and the dashed lines represent non-linear one-phase decay fit curves. 3.5. mRNA remains stable in NADES at room temperature for extended periods To evaluate the suitability of NADES as storage media for mRNA molecules and vaccines, the long-term stability of mRNA in NADES was evaluated. The mRNA translation in HeLa cells was evaluated after four months of storage at RT (21 °C). Results show that more than 50% of the mRNA translation was maintained after four months ( Figure 6 ). These results suggest that NADES can eliminate the need for refrigeration during the long-term storage of RNA molecules and RNA-based therapeutics. The ability to preserve such formulations at RT offers significant advantages for research, clinical applications, and public health. Download figure Open in new tab Figure 6. mRNA translation after long-term storage at room temperature (21°C) mCherry mRNA was stored in NADES for four months then transfected into HeLa cells. The percentage of fluorescent cells was quantified and normalized to transfection with control mRNA stored at −80 °C. Values are presented as means ± SD for n=3, and the dashed line represents non-linear one-phase decay fit curve. 4. Conclusions NADES exhibit low cytotoxicity in human cell models. Different NADES have varying effects on mRNA stability and function. NADES do not interfere with the mRNA in cellulo translation. NADES preserved over 50% of RNA functionality in cellulo for at least 48 h at 50 °C and 4 months at RT. Temperature fluctuations do not affect the stability of RNA stored in NADES. The biocompatibility of NADES positions them as strong candidates for biomedical applications, such as mRNA vacccines. NADES may extend the shelf life of mRNA vaccines and eliminate the need for refrigeration, substantially reducing storage and equipment costs. In vivo testing of mRNA stored in NADES should be carried out to evaluate potential immunogenicity of the mRNA-NADES formulations. Acknowledgment This work was funded by King Abdullah University of Science and Technology. A provisional patent related to this work has been filed (KAUST Ref#: 2025-069-01). Funder Information Declared King Abdullah University of Science and Technology, https://ror.org/01q3tbs38 , BAS/1/1087-01-01 References 1. ↵ Lesnik , E. A. & Freier , S. M. Relative Thermodynamic Stability of DNA, RNA, and DNA:RNA Hybrid Duplexes: Relationship with Base Composition and Structure . Biochemistry 34 , 10807 – 10815 ( 2002 ). OpenUrl 2. ↵ and, D.-M. Z. & Taira* , K. The Hydrolysis of RNA: From Theoretical Calculations to the Hammerhead Ribozyme-Mediated Cleavage of RNA . Chem Rev 98 , 991 – 1026 ( 1998 ). 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Water activity in biological systems - A review . Pol J Food Nutr Sci 62 , 5 – 13 ( 2012 ). OpenUrl View the discussion thread. Back to top Previous Next Posted July 26, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following NADES as Biocompatible Media for Thermally Stable RNA Molecules Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share NADES as Biocompatible Media for Thermally Stable RNA Molecules Lamya Al Fuhaid , Shahryar Khattak , Arwa Alghuneim , Imed Gallouzi , Young Hae Choi , Robert Verpoorte , Geert-Jan Witkamp , Andreia Farinha bioRxiv 2025.07.23.665770; doi: https://doi.org/10.1101/2025.07.23.665770 Share This Article: Copy Citation Tools NADES as Biocompatible Media for Thermally Stable RNA Molecules Lamya Al Fuhaid , Shahryar Khattak , Arwa Alghuneim , Imed Gallouzi , Young Hae Choi , Robert Verpoorte , Geert-Jan Witkamp , Andreia Farinha bioRxiv 2025.07.23.665770; doi: https://doi.org/10.1101/2025.07.23.665770 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Pharmacology and Toxicology Subject Areas All Articles Animal Behavior and Cognition (7619) Biochemistry (17642) Bioengineering (13865) Bioinformatics (41862) Biophysics (21409) Cancer Biology (18547) Cell Biology (25436) Clinical Trials (138) Developmental Biology (13358) Ecology (19863) Epidemiology (2067) Evolutionary Biology (24288) Genetics (15587) Genomics (22467) Immunology (17703) Microbiology (40301) Molecular Biology (17142) Neuroscience (88445) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4815) Physiology (7634) Plant Biology (15109) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9812) Zoology (2268)