Cloaking a molecule refers to the addition of a chemical modification to disguise or manipulate the molecule. Although this strategy originated in the early 1990s for control of small-molecule medicinal chemistry (Islam et al.), the importance of in vitro transcribed (IVT) mRNA vaccines and therapeutics has recently led to investigations of cloaking mRNA. Importantly, cloaking mRNA is carried out after conventional IVT using reversible chemistry to control uncloaking to fully functional mRNA. This blog discusses the first examples of mRNA cloaking, as reported by Prof. Eric Kool and his students at Stanford University (Fang et al. 2022), who used reporter mRNAs obtained from TriLink BioTechnologies.
The thermal and enzymatic instability of RNA present challenges to practical aspects of mRNA vaccines and therapeutics, imposing limits on mRNA analysis, storage, transportation, and pharmacology, as discussed elsewhere (Fabre et al. 2013). RNA degradation arises both from spontaneous thermal fragmentation and the action of ribonuclease enzymes, which are virtually ubiquitous. As depicted in Figure 1, the predominant mechanism of RNA thermal degradation starts with the attack of the deprotonated 2’-hydroxyl (red) on the adjacent 3’-phosphate, coordinated with the departure of the 5’-hydoxyl (blue), resulting in a 2’,3’-cyclic phosphate.
FIGURE 1. Mechanism of RNA thermal degradation. Taken from Fang et al. (2022) and free to use under a Creative Commons Attribution 4.0 International License.
According to Fang et al., developing strategies to preserve and subsequently restore RNA of any origin, post-synthetically under mild conditions, could constitute a major advance in RNA technologies in general. Such methodology would facilitate the storage and therapeutic use of a broad range of biologically active synthetic RNAs including IVT mRNA.
Based on prior findings by the Kool laboratory (Park et al. 2019), cloaking mRNA by acylation using acylimidazole reagents is attractive due to their ease of preparation, high water solubility, and effective 2’-acylation of RNA (Figure 2). Importantly, acylimidazoles can efficiently react with 2’-hydroxyls rather than exocyclic amines on nucleobases, and exhibit tunable hydrolytic half-lives, i.e., the 2’-carboxyl ester adducts can be reversed (uncloaked) by design to reinstate unmodified 2’-hydroxyl.
FIGURE 2. RNA cloaking using acylimidazole reagents with various substituents (R) to form 2’-carboxyl ester adducts. Taken from Fang et al. (2022) and free to use under a Creative Commons Attribution 4.0 International License.
Benchmarking mRNA Stabilization by Cloaking
To benchmark cloaking with acylimidazoles, Fang et al. used two different types of reporter mRNAs cloaked with the readily available acylimidazole reagent termed NAI-N3, known (Lee et al. 2022) to be selective for 2’-hydroxyls. First, they reacted NAI-N3 with TriLink’s CleanCap® enhanced green fluorescent protein (eGFP) mRNA (5-methoxyuridine). Reaction conditions pre-determined using a short (18 nt) synthetic RNA and MALDI-TOF mass spectrometry were applied to provide intermediate (~50%) and extensive (>90%) cloaking of the much longer (996 nt) eGFP-mRNA.
Capillary electrophoresis (CE) was then used to measure the lifespan of the fully intact uncloaked eGFP-mRNA versus that of eGFP-mRNA cloaked with NAI-N3. CE analysis of RNA fragments showed cloaking-dependent resistance to RNA degradation in water at 37°C. For instance, ~50% cloaking extended the lifespan of intact eGFP-mRNA by ~3-fold, while >90% cloaking was capable of near-complete shielding this mRNA from thermal cleavage, with <13% degradation after six days.
This NAI-N3 acylation-induced stabilization was further benchmarked with a second, even longer (1929 nt) mRNA, namely TriLink’s CleanCap® firefly luciferase (FLuc) mRNA (5-methoxyuridine). As with eGFP-mRNA, >90% cloaking almost completely blocked backbone cleavage of Fluc-mRNA over six days.
Fang et al. next explored whether the physico-chemical features of acylimidazole reagents can affect their abilities to suppress mRNA thermal degradation. A panel of six additional acylimidazole reagents containing structurally diverse substituents, R (Figure 2), were chosen to study structure-activity relationships. The six reagents were screened for modulation of mRNA thermal degradation by performing accelerated mRNA aging experiments in water at 37°C with eGFP-mRNAs each ~50% cloaked. Stabilization differences were not significant, indicating that the effect is independent of the size and aliphatic or aromatic nature of the studied R groups.
Cloaking RNA Suppresses Enzymatic Degradation by RNases and Biofluids
In principle, acylimidazoles can enhance enzymatic stability of mRNA by protecting phosphodiester linkages from enzymatic RNase-mediated attack by 2’-hydroxyls that is mechanistically similar to thermal degradation (Figure 1). Fang et al. therefore surveyed protective effects with representative RNases and biofluids to mimic the common enzymatic conditions mRNAs encounter during storage, handling, and biological applications.
As an initial assessment, they performed RNA degradation experiments with a synthetic model RNA (25 nt) that was >90% cloaked with the benchmarking acylimidazole NAI-N3. This cloaking effectively shielded cleavage sites on the model RNA from nucleolytic degradation by RNase A—the prototypical member of a superfamily of eight RNases abundant in vertebrate tissues, and RNase T1—a representative single-stranded RNA-cleaving endonuclease. In contrast, the unprotected model RNA was almost completely cleaved.
Consistent with the known short lifespan of RNA in blood (<15 seconds in human serum) (Tsui et al. 2002), the unprotected RNA was completely digested immediately upon contact with fetal bovine serum (FBS). In contrast, >90% cloaking with NAI-N3 blocked RNA degradation in both FBS and human cell (HeLa) lysates, suggesting possible uses in stabilizing mRNAs intracellularly.
Nucleophilic Reagents Remove 2’-Acylation to Uncloak mRNA
Nucleophiles are moieties with negative charge or unpaired electrons available for attacking electrophilic sites. Weakly basic nucleophiles such as pyridine are known to catalyze ester hydrolysis. To restore biological activity of mRNAs after cloaking, Fang et al. tested weakly basic nucleophiles as an uncloaking strategy to promote rapid hydrolysis of 2’-carboxyl esters (Figure 3).
FIGURE 3. RNA uncloaking using nucleophile (Nu:) reagents to catalyze rapid hydrolysis of 2’-carboxyl ester adducts. Taken from Fang et al. (2022) and free to use under a Creative Commons Attribution 4.0 International License.
Nucleophiles were screened against a short (18 nt) model RNA containing various types of acyl groups (R) at 37°C and neutral pH, which identified nine sensitive acylation-nucleophile pairs that promoted >50% removal of adducts within 2 hours. The next question was whether such uncloaking could be applied to cloaked mRNAs while keeping the strands intact. To address this critically important question, Fang et al. took advantage of the recent finding by the Kool lab (Park et al. 2021) that 2’-acylation can block translation when introduced into the coding region of mRNAs. Briefly, the widely used buffering agent tris(hydroxymethyl)aminomethane (Tris) was investigated as a nucleophile for uncloaking to restore translation of eGFP-mRNA cloaked with N,N-dimethylglycine (DMG), R = CH2N(CH3)2 (Figure 3).
Based on in vitro translation assays, ~50% cloaking with DMG strongly blocked the translation of eGFP-mRNA, while treatment with 50 mM Tris, pH=7.5 at 37°C led to 65±7% restoration of eGFP expression. CE analysis showed that the mRNA integrity remained largely unchanged. These data suggested that Tris alone could recover mRNA translation of adducts of DMG while keeping the mRNA intact.
mRNA Uncloaking in Human Cells Restores Translation with Extended Functional Half-Lives
Cells maintain high intracellular concentrations of the nucleophilic sulfur compounds cysteine and glutathione. The above-mentioned sensitivity of 2’-carboxyl esters towards nucleophilic hydrolysis (Figure 3) suggested to Fang et al. the possibility of spontaneous mRNA uncloaking by such bio-nucleophiles, leading to mRNA functional recovery. Transfection of eGFP-mRNA that was ~50% cloaked with DMG and analysis by fluorescence-activated cell sorting (FACS) were used to follow the appearance of the encoded eGFP reporter protein.
Green fluorescence was observed over two days, after an initial delay compared to uncloaked eGFP-mRNA. However, because eGFP protein is highly stable with a half-life >24 hours, protein degradation alone dominates expression and provides no information about mRNA functional half-lives. Fang et al. therefore used mRNA encoding the known (Kitsera et al. 2018) destabilized green fluorescent protein, d2GFP, wherein both mRNA and protein degradation occur with similar half-lives of ~2-3 hours.
Following liposomal transfection of HeLa cells, d2GFP-mRNA~50% cloaked with DMG led to extended (~10 hours) time of translation and 31% greater total protein output compared to uncloaked d2GFP-mRNA. While these initial findings are encouraging, future studies of other acyl R groups are needed to investigate the possibility of achieving more prolonged mRNA lifetimes and greater increases in protein outputs from cloaked mRNA.
Concluding Comments
The above findings by the Kool laboratory are the first demonstration of reversible cloaking of IVT mRNA to increase stability toward thermal or enzymatic degradation, and provide extended translation into a greater amount of encoded protein in cells. While this work was limited to initial exploration of structure-activity relationships using model mRNA reporter proteins and cell cultures, there are intriguing implications for future studies of cloaking mRNA vaccines and therapeutics.
For example, lipophilic alkyl-bearing cloaking reagents for mRNA might lead to improved encapsulation by lipid nanoparticles (LNPs) for delivery or, conceivably, cellular uptake without any additional transfection agents (Brown et al. 2022), i.e., self-transfecting cloaked mRNA. Also, cloaking reagents could install dyes for fluorescent detection of otherwise non-detectable mRNAs, as an alternative to conventional irreversible dye labeling. Cloaking circular RNA (circRNA) could possibly afford more protein production compared to uncloaked circRNA, which has outperformed traditional linear mRNA in antigen formation (Qu et al. 2022). Finally, mRNA control by non-chemical light-reversible acylation is a related avenue of exploration shown to be feasible (Velma et al. 2018).
In a broad sense, cloaking mRNA merges RNA chemistry, medicinal chemistry, and pharmacology into an exciting new interdisciplinary field with applications limited only by imagination.
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