Results
Overview
The present study aims to develop and optimize an aptazyme-based gene expression regulatory system for the sensing and response to a specific small molecule, S-adenosylmethionine (SAM), within cells. The ultimate objective is to engineer a smart genetic module, designated ReguSAMe, which responds to intracellular SAM levels. Through a negative feedback mechanism, this module is designed to achieve precise regulation of SAM concentration in hepatocytes.
Aptazyme-SAM Construction
Our Aptazyme-SAM was designed including two parts:ribozyme platform and SAM aptamer.
Construction of Guanine-Responsive Aptazyme
To evaluate the effectiveness of the HDV ribozyme1 and the hammerhead ribozyme2 as platforms for aptazyme engineering, we first tested the potential of two guanine-sensitive aptazymes, each built on one of these ribozyme backbones. The aptazymes were inserted into the 3' untranslated region (3' UTR) of an EGFP reporter gene mRNA. We transfected HEK293T cells with the following constructs: plasmid-EGFP-aptazyme-Gua-HDV, plasmid-EGFP-aptazyme-Gua-Hammer, and a control plasmid (plasmid-EGFP-BLANK) lacking a ribozyme insert. The experiment included two conditions: one in which the culture medium was replaced with DMEM containing 500 µM guanine eight hours post-transfection, and another where it was replaced with DMEM alone as a control. EGFP expression was observed using fluorescence microscopy 24 hours after transfection. The responsiveness of the aptazyme switches to guanine was assessed by the dynamic range of EGFP expression between low guanine (normal culture conditions) and high guanine (500 µM treatment) conditions. This dynamic range is defined as the ON/OFF ratio, calculated by dividing the fluorescence intensity in the active state (ON) by that in the inactive state (OFF).
The experimental results showed that the aptazyme-Gua-HDV exhibited a significantly higher ON/OFF ratio of 11.7-fold. In contrast, the aptazyme-Gua-Hammer showed a ratio of only 1.2-fold, indicating a much lower responsiveness to guanine compared to the HDV ribozyme scaffold. These results demonstrate that the HDV ribozyme scaffold is a superior platform, thereby providing a solid foundation for subsequent aptazyme engineering.
Selection of SAM Aptamers and Analysis of Fused Aptazyme Responsiveness
Selection of SAM Aptamers and Analysis of Fused Aptazyme ResponsivenessTo construct SAM-responsive aptazymes, we selected the aptamer domains from the structurally relatively simple SAM-I3 and SAM-VI4 riboswitches based on a survey of known SAM riboswitches and consultations with domain experts. These aptamers were fused to the HDV ribozyme by replacing the P4-L4 stem-loop of the ribozyme with the respective SAM-binding aptamer, resulting in the constructs Aptazyme SAM-I and Aptazyme SAM-VI-8. Each aptazyme was subsequently cloned into the 3' untranslated region (3' UTR) of an EGFP reporter gene, yielding the plasmids plasmid-EGFP-Aptazyme SAM-I and plasmid-EGFP-Aptazyme SAM-VI-8. These plasmids were then transfected into HEK293T cells. The functionality of the aptazymes was assessed under two culture conditions: DMEM supplemented with 500 µM SAM and SAM-free DMEM as a control.
EGFP expression analysis by fluorescence microscopy revealed that the Aptazyme-SAM I construct failed to exhibit self-cleavage activity under both the endogenous physiological SAM concentration in HEK293T cells and upon the addition of high exogenous SAM. In contrast, the Aptazyme-SAM VI aptazyme demonstrated self-cleavage activity under both conditions; however, it failed to display concentration-dependent responsiveness.
Consequently, we concluded that the SAM I riboswitch is unsuitable for constructing aptazymes via fusion with the HDV ribozyme. As for Aptazyme-SAM VI, although it displayed self-cleavage activity, the lack of concentration dependence suggests that its sensing threshold for SAM is likely lower than the normal physiological SAM concentration within HEK293T cells. This observation indicated the need to establish a cellular model with a gradient of SAM concentrations.
Literature review indicated that in normal mammalian cells, SAM synthesis is primarily catalyzed by the enzyme MAT2A. Cycloleucine acts as an inhibitor of MAT2A5.
We therefore decided to manipulate intracellular SAM levels by supplementing the culture medium with cycloleucine and SAM, aiming to create HEK293T cell models with low, normal, and high intracellular SAM concentrations.
Specifically, we used DMEM supplemented with 30 mM cycloleucine to downregulate intracellular SAM, standard DMEM to represent normal physiological SAM levels, and DMEM with 500 µM SAM to create a high-SAM condition. LC-MS analysis confirmed the efficacy of these treatments: compared to cells in normal DMEM, the intracellular SAM concentration in HEK293T cells treated with 30 mM cycloleucine decreased to 0.71-fold, while treatment with 500 µM SAM increased the intracellular concentration to 1.44-fold.
We next sought to evaluate the SAM responsiveness of the SAM VI-HDV aptazyme across the established cellular SAM concentration gradient. In parallel, we engineered an additional aptazyme by fusing the aptamer domain of the SAM-III riboswitch6, which shares structural similarities with the SAM-III riboswitch, to the HDV ribozyme.Evaluation of the aptazymes revealed distinct response profiles.
For Aptazyme-SAM VI, EGFP expression under low SAM conditions was significantly higher than that under both normal and high SAM conditions, yielding an ON/OFF ratio of 10.7-fold. In contrast, EGFP expression levels between the normal and high SAM conditions showed no significant difference. For Aptazyme-SAM III, no significant difference in EGFP expression was observed across the low, normal, and high SAM concentrations.
Evolution of SAM Concentration Response in Aptazymes:
Optimization of Linking Base Pairs between the SAM Aptamer and Ribozyme
To further optimize Aptazyme-SAM VI, we first modulated the number of base pairs linking the aptamer and the ribozyme. We constructed six variants of the SAM VI aptazyme, comprising linkers with 0, 2, 4, 6, 8, or 16 base pairs, each cloned into the EGFP reporter plasmid. These plasmids were transfected into the HEK293T cell model exhibiting the SAM concentration gradient (low, normal, high) as previously established. The SAM-responsive capability of each Aptazyme-SAM VI variant was subsequently assessed within this system.
The assessment of the Aptazyme-SAM VI variants revealed distinct functional profiles based on linker length. The variants Aptazyme-SAM VI-0, -2, and -4 showed no detectable self-cleavage activity under any of the SAM concentration conditions. In contrast, Aptazyme-SAM VI-6 and -16 exhibited constitutive self-cleavage, regardless of the intracellular SAM level. Consequently, Aptazyme-SAM VI-2, -4, -6, and -16 failed to demonstrate a concentration-dependent response to SAM. Notably, only Aptazyme-SAM VI-8 displayed a graded response to SAM, with EGFP fluorescence intensity decreasing progressively as the SAM concentration increased from low to high, yielding an ON/OFF ratio of 10.7.
In parallel, we optimized Aptazyme-SAM III by constructing four variants with linker lengths of 1, 2, 3, and 4 base pairs, each cloned into the EGFP reporter plasmid. These constructs were transfected into the HEK293T cell model with the established SAM concentration gradient to evaluate their SAM-responsive behavior.
Based on the experimental results, Aptazyme-SAM III-3 exhibited only a weak concentration-dependent response under normal and high SAM conditions, with an ON/OFF ratio of 1.2-fold, which was substantially lower than that of Aptazyme-SAM VI-8 (10.7-fold).
Consequently, Aptazyme-SAM VI-8 was selected for further optimization.
Optimization of Nucleotide Composition in Aptazyme-SAM VI-8
Following discussions with domain experts, we decided to incorporate the P0 stem7 of the SAM-VI riboswitch into the Aptazyme-SAM VI-8 construct, generating a new variant designated Aptazyme-SAM VI-P0. This construct was transfected into HEK293T cells with different intracellular SAM concentrations for validation. The results showed that the ON/OFF ratio for Aptazyme-SAM VI-P0 was 2.5-fold, compared to 9.4-fold for the original Aptazyme-SAM VI-8. The incorporation of the P0 stem into Aptazyme-SAM VI-8 did not improve the dynamic range but instead resulted in a significant reduction.
Based on the previous results, we decided to continue the optimization of Aptazyme-SAM VI-8. We systematically modulated the sequence variations of the base pairs linking the aptamer and the ribozyme, constructing and testing 20 distinct variants. In parallel, we collaborated with our dry-lab team to computationally predict the binding energy between these Aptazyme-SAM VI-8 variants and the SAM molecule.
The SAM-responsive capability of each variant was subsequently validated by transfecting the respective plasmids into the HEK293T cell model with the established SAM concentration gradient.
Based on our screening, we identified four top-performing aptazyme variants: Aptazyme-SAM VI-8 (ON/OFF ratio = 9.4-fold), Aptazyme-SAM VI-8-15 (7.5-fold), Aptazyme-SAM VI-8-16 (8.5-fold), and Aptazyme-SAM VI-8-20 (5.3-fold). All four variants demonstrated robust and favorable SAM-responsive activity.
We collectively designated these four engineered aptazymes as the "ReguSAMe" system. To achieve our ultimate therapeutic objective, we next sought to implement the ReguSAMe system within a cirrhotic cell model to construct a negative-feedback loop for precise SAM homeostasis.
Application of ReguSAMe in a Cirrhotic Cell Model
Establishment of an In Vitro Model of Liver Cirrhosis
We employed HepG2 cells to establish an in vitro model of liver cirrhosis. We first determined the relative expression levels of MAT1A and MAT2A. Analysis of MAT1A and MAT2A expression in HepG2 cells revealed that the expression level of MAT2A was significantly higher than that of MAT1A.
To establish the cirrhotic model groups, we treated HepG2 cells with DMEM supplemented with 60 mM or 30 mM cycloleucine (cLeu) to inhibit MAT2A enzyme activity. A control group was maintained in standard DMEM without treatment. Additional groups were treated with 0.5 mM or 1.0 mM SAM. Cells were harvested 24 hours post-treatment for liquid chromatography-mass spectrometry (LC-MS) analysis. The LC-MS results indicated that intracellular SAM concentration increased in the 60 mM and 30 mM cLeu-treated groups compared to the DMEM control group. Furthermore, SAM concentration in HepG2 cells treated with 0.5 mM and 1.0 mM SAM increased by 3.8-fold and 11.5-fold, respectively, compared to the DMEM control group.
The experimental results did not meet our expectations. Considering that cLeu itself is prone to degradation and may have been depleted by the 24-hour harvest time, and that MAT2A expression might have been feedback-upregulated, catalyzing SAM synthesis and leading to the increased SAM concentration at 24 hours, we set five different treatment time points with DMEM + 30 mM cLeu: 2, 6, 10, 16, and 32 hours. The results from qPCR validation of MAT2A mRNA abundance indicated that 2 hours was the optimal treatment time.
We confirmed via LC-MS that the intracellular SAM concentration in the 2-hour DMEM + 30 mM cLeu treatment group was reduced to 26% compared to the DMEM-only control group. Based on this confirmation, the cirrhotic cell model was successfully established.
Plasmid-EGFP-Aptazyme-SAM VI-8, plasmid-EGFP-Aptazyme-SAM VI-8-15, plasmid-EGFP-Aptazyme-SAM VI-8-16, and plasmid-EGFP-Aptazyme-SAM VI-8-20 were transfected into both the established cirrhotic cell model and normal hepatocytes to validate the SAM-responsive capability of the ReguSAMe system.
Aptazyme-SAM VI-8 exhibited an ON/OFF ratio of 3.1-fold, Aptazyme-SAM VI-8-15 exhibited an ON/OFF ratio of 3.1-fold, Aptazyme-SAM VI-8-16 exhibited an ON/OFF ratio of 2.6-fold, and Aptazyme-SAM VI-8-20 exhibited an ON/OFF ratio of 1.9-fold. Concurrently, flow cytometry was performed to further validate the reliability of our components. The experimental results were consistent with the observations under fluorescence microscopy, with the three ReguSAMe constructs demonstrating a response to SAM.
Construction of a Negative Feedback Circuit for SAM Regulation
We replaced the reporter gene EGFP with MAT1A to construct a SAM concentration-responsive negative feedback circuit. The constructs plasmid-MAT1A-Aptazyme-SAM VI-8, plasmid-MAT1A-Aptazyme-SAM VI-8-15, plasmid-MAT1A-Aptazyme-SAM VI-8-16, and the control plasmid-MAT1A-blank were transfected into the cirrhotic cell model to validate MAT1A mRNA levels.
During the 16h to 48h period, compared to the control group, ReguSAMe successfully maintained intracellular MAT1A at a consistently higher level without causing overexpression as observed in the BLANK group. The negative feedback pathway was successfully constructed, and we decided to apply it to a therapeutically viable mRNA platform.
LNP Preparation
To apply ReguSAMe in mRNA therapeutics, our primary step involves constructing a suitable carrier for mRNA delivery. Lipid nanoparticles (LNP) are one of the most widely used delivery systems in today's gene drugs, mainly for liver delivery and vaccine applications.
We selected DLin-MC3-DMA as the foundational cationic lipid due to its ionizable nature. Under acidic conditions, it becomes protonated and binds to negatively charged mRNA, thereby promoting nanoparticle formation and membrane fusion. To further optimize the LNP properties, distearoylphosphatidylcholine (DSPC) was incorporated to modulate bilayer fluidity and enhance cellular uptake, while cholesterol was included to fill interstitial voids between lipids and improve structural integrity. We encapsulated mRNA into LNP by manual mixing method.
The prepared LNPs were characterized for their size distribution, which was found to be concentrated within the range of 80–120 nm. This narrow size distribution confirms the successful preparation of our LNP formulation.
Validation of mRNA Cleavage Efficiency
To evaluate the feasibility of ReguSAMe in mRNA-based therapeutic systems, we constructed EGFP mRNA with Aptazyme SAM-VI-8, SAM-VI-8-15, and SAM-VI-8-16 incorporated into the 3'UTR region. The ReguSAMe-containing mRNA was obtained by in vitro co-transcription, encapsulated into LNP, and delivered into cirrhotic cell models and normal SAM concentration cell models, respectively. Fluorescence signals were captured using fluorescence microscopy, and the ratio of their intensities was used as an indicator to assess the concentration-responsive efficiency of ReguSAMe.
The results showed that the fluorescence intensities of Aptazyme SAM-VI-8-16 and SAM-VI-8-15 under low SAM concentrations were significantly higher than those under normal physiological SAM concentrations. The experiment preliminarily demonstrates that our aptazyme components can undergo self-cleavage in response to normal physiological SAM concentrations.
To mitigate experimental artifacts arising from variations in cell numbers across experimental groups, we further characterized the system using flow cytometry. The results obtained were consistent with fluorescence microscopy observations, confirming that Aptazyme SAM-VI-8-16 and SAM-VI-8-15 exhibited the most significant SAM responsiveness.
Validation of the mRNA Therapeutic System
To validate that ReguSAMe can restore SAM concentration to the physiological level, we replaced the EGFP coding sequence (CDS) in the mRNA with the gene encoding MAT1A enzyme. MAT1A mRNAs equipped with Aptazyme SAM-VI, SAM-VI-8-15, or SAM-VI-8-16 were delivered into both cirrhotic cell models and cells with normal physiological SAM concentrations.
If our system functions as designed, under low SAM conditions in the cirrhotic model, ReguSAMe remains intact, allowing MAT1A expression and subsequent SAM synthesis. As SAM concentration rises, ReguSAMe senses the increase and undergoes self-cleavage, leading to rapid degradation of the mRNA. To maintain SAM within the normal physiological range, the remaining mRNA levels should eventually stabilize at comparable concentrations in both cirrhotic and normal SAM concentration models.
The experimental results confirmed our hypothesis. Within the first 3 hours, the cleavage rate of ReguSAMe was lower in the cirrhotic model than in the normal SAM model. After SAM levels were restored to normal via MAT1A-mediated synthesis, the remaining mRNA levels became comparable between the two groups, demonstrating that ReguSAMe maintains SAM concentration within a stable, physiological range.
Based on the qPCR results, we confirmed that Aptazyme SAM VI-8-15 exhibited the most rapid response and maintained a cleavage rate closest to that of the DMEM group after SAM concentration was restored.
We therefore proceeded to validate the SAM concentration restoration capability of Aptazyme SAM-VI-8-15 by direct SAM quantification. MAT1A mRNA constructs with and without the Aptazyme SAM-VI-8-15 element were delivered into cirrhotic cell models. To control for potential effects of the mRNA delivery process itself on cellular state, EGFP mRNA was delivered in parallel. Cells maintained in untreated DMEM served as the normal physiological SAM concentration control, providing a baseline to confirm successful SAM restoration.
The optimal sampling time point for LC-MS was determined based on previous qPCR data, which indicated comparable remaining mRNA levels between groups at approximately 6 hours—suggesting that SAM concentration had been restored to the physiological range by this time. Accordingly, mass spectrometry analysis was performed at the 6-hour time point.
The experimental results demonstrated that delivery of MAT1A mRNA without the Aptazyme SAM-VI-8-15 element led to a significant upregulation of SAM concentration, substantially exceeding the normal physiological level. In contrast, MAT1A mRNA incorporating the Aptazyme SAM-VI-8-15 element restored SAM concentration to approximately twice the baseline level.
However, as these data represent only a single time point, we aim to investigate the temporal dynamics of SAM concentration in future studies. We anticipate that over time, SAM concentration may stabilize within the normal physiological range.