iGEM 2025 — Team dut-china

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## A Detailed Record of Our Project

  Embark on a journey through our Wet Lab Notebooks, where every experiment is documented step-by-step. These comprehensive PDFs capture not just our protocols and results, but the very insights that shaped our project. Dive in and explore the process behind the science!

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Overview

Drug target selection cycle
▶

Cycle 1

Cycle 2

Cycle 3

Cycle 4

UTR optimization loop
▶

Cycle 1

Cycle 2

Cycle 3

Switch cycle
▶

Cycle 1

Cycle 2

Cycle 3

Cycle 4

Expression vector cycle
▶

Cycle 1

Cycle 2

References

# Overview

This project systematically promotes the development of mRNA drug platforms through multiple parallel and interrelated engineering cycles: at the design level, we conceived a multi-level regulatory blueprint including target degradation, protein expression, UTR optimization, molecular switching, and targeted delivery systems; In the construction stage, we used plasmid vectors and in vitro transcription techniques to successfully construct various functional mRNA molecules and surface display extracellular vesicles. During the testing phase, we used various methods such as RT-qPCR, Western Blot, fluorescence detection, and nanoflow cytometry to rigorously evaluate the functional efficiency of each component. Finally, in the learning phase, we conduct in-depth analysis based on the experimental data of each cycle (such as efficient siRNA silencing, UTR sequence effect differences, leakage expression problems of switches, etc.) , and then drive the next round of more targeted optimization design (such as integrated MFE optimization) . UTR model, exploring switches with different CAG repeats, etc. and gradually transformed the initial concept into an integrated drug platform with increasingly perfect functions and more accurate regulation.

# Drug Target Selection Cycle

## Cycle 1

D
Design

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that plays a complex dual role in liver physiology and pathology. In hepatocellular carcinoma (HCC) , TGF-β protein inhibits tumors in the early stages and strongly promotes their malignant progression in the late stages. Here, we select TGF-β protein as our protein degradation target. In order to verify that the protein expression of this target can be artificially regulated, we designed siRNAs targeting TGF-β protein mRNA. It is hoped that RNA interference technology can be used to silence TGF-β protein gene expression from the RNA level.

B
Build

We designed siRNAs targeting the 48th, 339th, and 642nd bases of TGF-β protein mRNA, respectively , the prepared siRNA was transfected into Hepg cell lines by lipid nanoparticles (LNPs) , and the gene silencing effect was tested after 48h of culture.

T
Test

The cells were collected, RNA was extracted, and the mRNA expression level of TGF-β gene was detected by RT-qPCR. After cell lysis, Western blot experiments were performed to test the expression level of target genes from the protein content level.

![Figure 1: Analysis of RT-qPCR results from TGF-β gene siRNA silencing experiments](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle1.webp)

Figure 1. Analysis of RT-qPCR results from TGF-β gene siRNA silencing experiments. Data from three groups (siRNA1, siRNA2, siRNA3) were obtained from samples transfected with siRNAs targeting positions 642, 48, and 339 of the TGF-β mRNA, respectively. Quantitative PCR was performed on a real-time PCR system for data collection and analysis. All samples were prepared and replicated three times. The experiment was performed independently at least three times. To calculate relative concentrations, CT values were obtained for all samples. ΔCt represents a normalized, relative gene expression level. This normalization was achieved by standardizing the degradation effect of TGF-β siRNA. Normalized expression for each sample was obtained by subtracting the CT value of the internal reference gene (GAPDH) from the same sample, designated as ΔCt. Compare the ΔCt of the experimental group with the ΔCt of the control group. Formula: ΔΔCt = ΔCt(siRNA) individual - ΔCt(siRNA) mean. Then convert this value to a comparative fold change by performing log(2 −(ΔΔCT)). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

![Figure 2: Western Blot results for TGFβ gene silencing by siRNA](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle2.webp)

Figure 2. Western Blot results for TGFβ gene silencing by siRNA. Lanes siRNA1, siRNA2, and siRNA3 correspond to samples transfected with siRNA targeting positions 642, 48, and 339 of TGFβ mRNA, respectively. Lanes Untreated1–Untreated3 on the right serve as blank controls. Cells were lysed and analyzed 48 hours after siRNA transfection. Protein loading was 20 μg.

![Figure 3: Western Blot Gray Value Analysis of TGFβ Gene Silencing by siRNA](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle3.webp)

Figure 3. Western Blot Gray Value Analysis of TGFβ Gene Silencing by siRNA. Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

Based on the RT-qPCR experimental results, the degradation rate of the three siRNAs of TGF-β reached 70% or more, and at the same time Western Blot experimental grayscale analysis also showed that we successfully silenced the TGF-β gene. It was proved that the protein expression of this target can be artificially regulated, laying the foundation for our subsequent experimental design.

## Cycle 2

D
Design

Based on the verification that the expression of TGF-β protein can be artificially regulated, we designed mRNA with a protein degradation module to degrade TGF-β (TGF-β degradation RNA) to reduce the expression of this gene at the protein level. The element consists of three modules, namely the Trim21 protein, the ligation peptide, and a short peptide that can specifically bind to TGF β proteins. The fusion protein composed of these three modules can specifically bind to TGF-β protein and reduce the content of TGF-β protein by mediating the ubiquitination degradation pathway. We plan to deliver this mRNA into cells to verify the effectiveness of our design by detecting changes in the content of TGF-β protein.

B
Build

We used the pUC57 plasmid as a vector to design expression plasmids capable of characterizing target RNA. For TGF-β degradation RNA plasmids, the target sequences include: short peptide + ligation peptide + trim21 sequence targeting TGF β protein. We also integrate the T7 promoter into plasmids for in vitro transcription of TGF-β degradation RNA.

![Figure 4: TGF-β degradation RNA demonstration platform](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle4.webp)

Figure 4. TGF-β degradation RNA demonstration platform

T
Test

After constructing the plasmid, we linearized the expression vector and performed T7 RNA polymerase-mediated RNA transcription in vitro, but during the experiment, we found that the RNA concentration of the transcript product was low, making it difficult to proceed to the next experiment.

L
Learn

After reviewing the literature and researching multiple sources, we decided to replace the T7 promoter with a more reliable one, hoping to obtain a higher concentration of RNA products for subsequent validation experiments, while providing a guarantee for RNA transcription in other module experiments.

## Cycle 3

D
Design

Further, we designed a protein regulatory system with both components, and the two regulatory elements are linked by P2A peptides. It is hoped that the expression of tumor suppressor gene-related proteins can be improved while degrading the proteins expressed by the original oncogene in hepatocellular carcinoma cells, thereby improving the anti-cancer effect of drugs.
In order to verify the feasibility of upregulating and downregulating two different target proteins at the same time, we plan to first introduce eGFP-expressing mRNAs into cells, and then target eGFP mRNA, siRNA and mRNA expressing Luciferase were delivered into cells, and the feasibility of this strategy was verified by detecting changes in fluorescence intensity before and after cell transfection.

![Figure 5: Co-delivery experiment of eGFP siRNA and Luc mRNA](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle5.webp)

Figure 5. Co-delivery experiment of eGFP siRNA and Luc mRNA

B
Build

We designed siRNAs targeting eGFP fluorescent proteins (eGFP siRNAs) and those that can stably express eGFP/Luciferase, respectively mRNA (eGFP mRNA and Luc mRNA) . The eGFP mRNA was transfected into 293T cells first, and then the eGFP siRNA was combined with the Luc mRNA. The feasibility of this strategy was verified by detecting the fluorescence intensity of cells after 24h of transfection.

T
Test

The validation experiment was performed using the 293T cell line, and the control group was transfected with only eGFP mRNA and Luc mRNA .The remaining groups were introduced with Luc RNA + eGFP RNA + eGFP siRNA respectively, and another was set up Blank control group. Fluorescence detection was performed after 48h of transfection.

![Figure 6: Fluorescence detection results of eGFP siRNA and Luc mRNA co-delivery experiment](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle6.webp)

Figure 6: Fluorescence detection results of eGFP siRNA and Luc mRNA co-delivery experiment a: EGFP fluorescence intensity detected by flow cytometry using the B525 channel, presented as Mean Fluorescence Intensity (MFI) b: Fluorescence intensity measured at 560 nm wavelength using a microplate reader, presented as Relative Light Units (RLU). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

The experimental results showed that the green fluorescence intensity of the experimental group transfected with eGFP siRNA was significantly different from that of the control group without transfection of siRNA (p<0.0001) and was treated in the same experiment .There was no significant difference in fluorescence intensity between the two groups at 560nm wavelength (p>0.05) , indicating that we successfully expressed eGFP while using siRNA to reduce expression Luciferase proved the feasibility of up-regulating and down-regulating two different target proteins at the same time, laying the foundation for further experimental exploration.

## Cycle 4

D
Design

In order to evaluate the application potential of the drug, firstly, we plan to use siRNA to simultaneously silence the expression of two different fluorescent proteins, and verify the feasibility of down-regulating the expression of multiple proteins at the same time by detecting fluorescence intensity. We expect this drug to complement as a molecularly targeted drug in combination therapy and to be used in combination with antibody drugs targeting key immune checkpoints (e.g., PD-1/PD-L1 axis) , as well as targeting tumor angiogenesis (eg. VEGF/VEGFR signaling pathway) to reverse the immunosuppressive microenvironment and inhibit tumor progression through a multi-mechanism combination, thereby improving the overall therapeutic effect of advanced liver cancer.

![Figure 7: Co-delivery degradation experiment of eGFP siRNA and Luc siRNA](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle7.webp)

Figure 7. Co-delivery degradation experiment of eGFP siRNA and Luc siRNA

B
Build

We designed siRNAs targeting eGFP fluorescent proteins/Luciferase (eGFP siRNA and Luc mRNA, respectively) and mRNAs that stably express eGFP/Luciferase (eGFP mRNA and Luc mRNA, respectively ) . The eGFP mRNA and Luc mRNA were transfected into the cells first, and then the eGFP siRNA and Luc siRNA were transfected Co-delivered into cells to verify the feasibility of the strategy by detecting changes in fluorescence intensity before and after cell transfection.

T
Test

The 293T cell line was used for validation assays, and the control group was transfected with eGFP siRNA and Luc mRNA, and the experimental group was transfected with Luc RNA + eGFP RNA + eGFP siRNA +Luc siRNA, and a blank control group. Fluorescence detection was performed after 48h of transfection.

![Figure 8: Fluorescence detection results of eGFP siRNA and Luc siRNA co-delivery degradation experiment](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle8.webp)

Figure 8: Fluorescence detection results of eGFP siRNA and Luc siRNA co-delivery degradation experiment a: EGFP fluorescence intensity detected by flow cytometry using the B525 channel, presented as Mean Fluorescence Intensity (MFI) b: Fluorescence intensity measured at 560 nm wavelength using a microplate reader, presented as Relative Light Units (RLU). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

The experimental results showed that the green fluorescence intensity of the experimental group transfected with eGFP siRNA and Luc siRNA was significantly different from that of the control group without transfected siRNA (p<0.0001) Under the same experimental treatment, there was a significant difference in fluorescence intensity between the two groups at 560nm wavelength (p<0.001) , indicating that we used it eGFP siRNA and Luc siRNA successfully reduced both eGFP and Luciferase proved the feasibility of our strategy of down-regulating the expression of multiple proteins at the same time, and laid the foundation for further experimental exploration.

# UTR Optimization Loop

## Cycle 1

D
Design

In order to obtain the manually optimized design of the 5’UTR sequence, we selected two models, UTRGAN and UTR-Insight, and built the basis based on the model characteristics and operation requirements. The computing environment of the PyTorch framework is configured with GPU acceleration resources to meet the computing power requirements of deep neural network training, and the version conflict problem of the two model dependencies is solved through virtual environment isolation – UTRGAN. The required WGAN-GP optimization module, transpose convolution implementation component, and UTR-Insight dependent UTR-LM pre-trained language model. The Conv-Former architecture-related tools are deployed in separate virtual environments to ensure that their functions are functioning properly.
Based on these two independent models, we generated the first UTR sequences. After UTR-Insight prediction and screening, 10 high MRL sequences were selected for the next experimental verification. We selected Luciferase as the reporter protein and designed an RNA capable of Luciferase expression (Luc-UTR RNA) . To evaluate the regulatory efficiency of different 5’UTR variants.

B
Build

We used the pUC57 plasmid as a vector to construct an expression vector for evaluating the efficiency of 5’UTR. A Luciferase coding sequence was inserted into the plasmid as a reporter gene, and the T7 promoter was integrated to enable in vitro transcriptional synthesis of Luc-UTR RNA. We designed 10 pairs of specific primers for the UTR sequence output of the model, integrated the designed sequence into the expression vector by PCR, and obtained the corresponding artificial 5'UTR and LUC-UTR RNA by in vitro transcription technology.

![Figure 9: Luc-UTR RNA Display Platform](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle9.webp)

Figure 9. Luc-UTR RNA Display Platform

T
Test

The prepared mRNA was transfected into a 293T cell line by lipid nanoparticles (LNPs) , cultured for 24h and then lysed the cells, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.

![Figure 10: Characterization of Regulatory Efficiency for the First Set of 5'UTR Sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle10.webp)

Figure 10. Characterization of Regulatory Efficiency for the First Set of 5'UTR Sequences Sequence numbers 1–10 represent nine differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, while NC serves as a blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).

L
Learn

Through the analysis of wet experimental data, we found that there is still a big gap between the regulatory ability of the 5’UTR generated by the existing model and the existing artificially synthesized high-expression 5’UTR sequences, generate the model for further improvement and optimization.

## Cycle 2

D
Design

Based on the experimental results of cycle1, we have improved the generative model. The core constraint system that integrates the minimum free energy (MFE) prediction ability and systematically incorporates the key regulatory dimension of mRNA stability into the sequence design is achieved through deep coupling of the original model prediction link. Simultaneous prediction - the stability of the secondary structure can be quantified in real time during the sequence generation process, and the risks such as strong stem-ring structure caused by energy anomalies can be identified in advance, so as to solve the experimental problems caused by stability imbalance in the first batch of sequences. At the same time, we add a gradient optimization mechanism, based on the original generation logic of UTRGAN, and dynamically guide the parameter space generated by the sequence through the gradient optimization algorithm, hoping to improve the effect of the sequence.
We use the self-designed fusion model to optimize the design of the sequence, calculate the minimum free energy of the resulting sequences, and select the second batch of 15 sequences for wet experimental verification after considering MRL and MFE.

B
Build

To test the expression efficiency of the 5’UTR sequences we generated, while unifying our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle1, designed 15 pairs of specific primers for the 15 UTR sequences output by the fusion model, and integrated the designed sequences into the expression vector by PCR. mRNA with the corresponding artificial 5’UTR was obtained by in vitro transcription technology.

T
Test

The prepared mRNA was transfected into a 293T cell line by lipid nanoparticles (LNPs) , cultured for 24h and then lysed the cells, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.

![Figure 11 (Part 1): Efficiency Characterization of the Second Set of 5'UTR Sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle11-1.webp)
![Figure 11 (Part 2): Efficiency Characterization of the Second Set of 5'UTR Sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle11-2.webp)

Figure 11. Efficiency Characterization of the Second Set of 5'UTR Sequences Sequence numbers 11-24 represent 14 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, while NC serves as a blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).

L
Learn

Compared to the reference sequence, we designed the sequence with low expression. We analyze the reasons for the results of the new round of experiments: (1) In the actual reproduction of the UTR-Insight model, we did not perform the key pre-training steps and directly used the randomly initialized transformer. The encoder is connected to the main training of the model. This omission led to the encoder not being able to effectively capture the hierarchical biological features of the sequence, ultimately resulting in the model's prediction accuracy failing to reach the level in the literature. (2) Due to too many changes to the code during the use of the model, the model effect is affected. (3) There is no reference for control, and only the sequence optimized by the model random design is used for numerical screening.

## Cycle 3

D
Design

In response to the problems encountered in the first two loops, we further improved the model by retraining the model and designing an automated workflow to optimize the design of the sequence by running the two models in parallel. The MRL scores of UTR sequences with known high efficiency were calculated, and the sequences with the same level or higher scores were screened, and the above sequences were compared in terms of MFE and CG content.
We designed the third batch of sequences with the new model and selected 14 sequences for the next experimental verification. In order to further characterize our 5’UTR effect, we collected NCA-7d-5'UTR sequences and Albumin-5'UTR. The former is a highly expressed endogenous 5’UTR, and the latter is an albumin gene-derived 5’UTR, both of which are naturally occurring 5’UTR sequences in nature. It has been used in mRNA therapy research. We want to compare it with the regulation effect of our self-designed 5’UTR to better evaluate our optimization results.

B
Build

To test the expression efficiency of the 5’UTR sequences we generated, while unifying our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle1, designed 15 pairs of specific primers for the 15 UTR sequences output by the fusion model, and integrated the designed sequences into the expression vector by PCR. mRNA with the corresponding artificial 5’UTR was obtained by in vitro transcription technology.

T
Test

The prepared mRNA was transfected into a 293T cell line by lipid nanoparticles (LNPs) , cultured for 24h and then lysed the cells, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.

![Figure 12: The third batch of 5'UTR sequence regulatory efficiency characterization](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle12a.webp)

Figure 12. The third batch of 5'UTR sequence regulatory efficiency characterization. Numbers 26-39 represent 14 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized sequence, the NCA group uses the NCA-7d-5'UTR sequence, the Alb group uses the Albumin-5'UTR sequence, and NC serves as the blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).

![Figure 13: Comparative Characterization of Regulatory Efficiency Among Three Batches of 5'UTR Sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle13a.webp)

Figure 13. Comparative Characterization of Regulatory Efficiency Among Three Batches of 5'UTR Sequences. Numbers 1–39 denote 34 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, the NCA group uses the NCA-7d-5'UTR sequence, the Alb group uses the Albumin-5'UTR sequence, and NC represents the blank control without transfection of RNA. Fluorescence intensity data were measured at 560 nm wavelength using a microplate reader. The vertical axis represents the ratio of fluorescence intensity in the experimental group to that in the PC group.

![Figure 14: Comparison of Output Quality Among Three Batches of 5'UTR Models](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle14a.webp)

Figure 14. Comparison of Output Quality Among Three Batches of 5'UTR Models Fluorescence intensity data were measured at 560 nm wavelength using a microplate reader. The vertical axis represents the ratio of fluorescence intensity in the experimental group to that in the PC group.

L
Learn

According to the experimental data analysis, the design quality of the third batch of 5’UTR was improved compared with the previous two batches, and the regulatory effect of the 5’UTR sequence No. 37 was better than that of the two high expressions in nature 5’UTR, with application potential. So far, the iteration of our UTR optimization model has achieved certain results.

# Switch Cycle

## Cycle 1

D
Design

The Toehold switch is a modularly designed cis-acting riboswitch that functions to regulate the translation of downstream genes by sequence-specific binding to specific trigger RNAs. Its composition includes a switch element located in the untranslated region of the 5' end of the mRNA, which spontaneously folds into a hairpin secondary structure, thereby hiding the ribosome binding site and inhibiting translation initiation; When fully complementary trigger RNAs bind to this region through a strand replacement reaction, they cause switch conformational rearrangements, exposing ribosome binding sites that specifically activate protein expression of the gene of interest.

![Figure 15: Regulatory Mechanism of the Toehold Switch](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle15.webp)

Figure 15.Regulatory Mechanism of the Toehold Switch

At the beginning of the project, we found a toehold switch sequence with microRNA as an aptamer that can be used in eukaryotic systems through literature research, and hoped to optimize it and apply it to our drug platform system to improve the cell selectivity of our drugs. For this sequence, we designed an mRNA (t-T-mRNA) with a toehold switch sequence and a Luciferase sequence. The trigger RNA in the switch is miR-21.

B
Build

In order to test the expression regulation efficiency of the toehold switch sequences collected by us, we used pUC57 plasmid as a carrier to construct an expression vector for evaluating the expression regulation efficiency of molecular switching. Here we use a toehold switch whose trigger RNA is miR-21. The Luciferase coding sequence is inserted into the plasmid as a reporter gene, and the T7 promoter is integrated for in vitro transcription and RNA synthesis. We designed a pair of specific primers for the artificially designed toehold sequence, and the switch sequence was integrated into the vector by PCR 5’ UTR behind the RNA sequence, mRNA with corresponding switch sequences (t-T mRNA) was obtained by in vitro transcription technology.

T
Test

The prepared t-T RNA and miR-21 were transfected into a 293T cell line by lipid nanoparticles (LNPs) , and the cells were lysed after 24h of culture, and Luciferase substrate was added. The fluorescence intensity was tested within 2h.

![Figure 16: Validation of toehold sequence regulation on protein expression](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle16a.webp)

Figure 16. Validation of toehold sequence regulation on protein expression Luciferase activity was measured and presented as Relative Light Units (RLU). Group T1 was transfected with t-T mRNA only; Group T2 was transfected with t-T mRNA and miR-21 mimics; Group T3 was transfected with t-T mRNA and miR-21 inhibitor; The NC group received no RNA transfection. Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

The experimental results showed that there was a significant difference in the fluorescence intensity measured at 560 nm between the control group transfected with t-T-mRNA and the experimental group transfected with t-T-mRNA and miR-21 at 560 nm (p<0.0001) , indicating that the toehold sequence has a regulatory effect. However, there is still room for optimization at the opening level, so we decided to try to generate and optimize some toehold sequences for subsequent experimental validation.

## Cycle 2

D
Design

To solve the problem of low level of openness, we studied the special structure of the eukaryotic. Toehold sequence and other factors that affect its function, such as Kozak offset, toe pairing, GC content, etc. Two sequences were manually designed for dry experimental verification, and it was found that the characterization efficiency was too low and the results were not ideal.
Next, we took the Tau team's Triggate project in 2022 to try out switch designs using their platform, which we obtained 25. The Toehold switch sequence of the eukaryotic system was based on the next experimental verification.

B
Build

To test the expression efficiency of the Toehold switches we generated, and to harmonize our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle1 and designed 25 pairs of specific primers for the 25 UTR sequences output by the triggate platform . For specific primers, the corresponding mRNAs are t-A mRNA to t-Z mRNA. The designed sequences were integrated into the expression vector by PCR, and mRNA with the corresponding artificial Toehold switch sequence was obtained by in vitro transcription technology.

T
Test

The obtained t-C mRNA, t-E mRNA, t-I mRNA, t-K mRNA will be prepared and cycle1 prepared t-T-mRNA and miR-21 transfected to 293T by lipid nanoparticles (LNPs) , respectively. In the cell line, the cells were lysed after 24h of culture, Luciferase substrate was added, and the fluorescence intensity was tested within 2h.

![Figure 17: Validation of the self-designed toehold sequence for regulating protein expression](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle17.webp)

Figure 17. Validation of the self-designed toehold sequence for regulating protein expression. Luciferase activity was measured and presented as Relative Light Units (RLU). Groups C1, E1, I1, K1, and T1 were transfected with mRNA containing only the toehold switch sequence and Luciferase sequence. Groups C2, E2, I2, K2, and T2 were transfected with mRNA and miR-21 mimics. Groups C3, E3, I3, K3, and T3 were transfected with mRNA and miR-21 inhibitors. The PC group was transfected with mRNA expressing Luciferase without the switch sequence, while the NC group received no transfection. Statistical significance was assessed using ANOVA with Bonferroni-corrected multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

The experimental results show that the fluorescence expression of the self-designed toehold switch is greatly increased compared with the sequence in cycle1, but the leakage expression is higher at the same time, and the regulatory effect is poor. Switch generation models while exploring other feasible molecular switch designs.

## Cycle 3

D
Design

We set our sights on other types of RNA molecular switches. After investigation, we found an RNA-ON switch based on exCAG sequences. This switch consists of a miR-21 binding region and a repressive sequence, located downstream of the mRNA polyA tail. The repression region consists of repeated CAG sequences that silence the ORF region by inducing RNA gelation. When the concentration of miR-21 in the cell is high, its complementary region pairs with miR-21 and recruits the RISC complex to cleave the inhibitory sequence, thereby degelating the RNA and making the ORF normal expression for cell selectivity.

![Figure 18: Regulatory Mechanism of the exCAG Switch](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle18a.webp)

Figure 18. Regulatory Mechanism of the exCAG Switch

Based on this principle, we designed an mRNA (CAG30 mRNA) with the mCherry sequence as the reporter gene. The posterior insertion of the polyA tail of RNA adds a miR-21 binding sequence and a 30x repeated CAG sequence as an inhibitory sequence. The trigger RNA switched in this mRNA is miR-21.

B
Build

In order to test the expression regulation efficiency of the exCAG switch sequence we designed, we designed and constructed an expression vector using the pUC57 plasmid as a carrier. The mCherry coding sequence was inserted into the plasmid as a reporter, and the miR-21 binding sequence was added sequentially after the polyA tail, and 30× Repeated CAG sequences and integration of the T7 promoter upstream of the 5’UTR for in vitro transcriptional synthesis. CAG30 mRNA with corresponding switch sequences was obtained by in vitro transcription.

T
Test

The prepared CAG30 mRNA and miR-21 were transfected into a 293T cell line by lipid nanoparticles (LNPs) and cultured for 24 h and then by flow cytometry B610 channel detects fluorescence signals.

![Figure 19: Fluorescence detection results of mCherry expression following 30xCAG sequence regulation](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle19.webp)

Figure 19. Fluorescence detection results of mCherry expression following 30xCAG sequence regulation. mCherry fluorescence was detected by flow cytometry and presented as Mean Fluorescence Intensity (MFI). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

The experimental results showed that the experimental group transfected with CAG30 mRNA and miR-21 inhibitor was the same as that transfected with CAG30 mRNA and miR-21 mimic. There was no significant difference in the fluorescence intensity of mCherry detected by B610 channel by flow cytometry (p>0.05). The switch design and control effect is not good, and there is still room for optimization. Therefore, we decided to explore the optimal number of replicates of exCAG in the inhibition sequence to increase the switch-induced fold and the level of the open state.

## Cycle 4

D
Design

In order to further optimize the switch design, increase the induction factor and open state level, and reduce the impact on normal cells, and explore the lowest CAG repeats, we designed two sets of switches with the number of CAG repeats of 3× and 10× for further experimental verification. We speculate that the optimal repeat may be between 3× and 30×, and hope to further narrow the range of sequence screening through this experiment, and finally find the optimal sequence through engineering cycle iteration.
We designed two mRNAs with mCherry sequences as reporters (CAG3 mRNA and CAG10 mRNA) , and miR-21 was added to the posterior insertion of the polyA tail of both RNAs. The sequences were combined, and two exCAG sequences with 3× and 10× repeats were inserted as inhibitory sequences, respectively. The trigger RNA switched in this mRNA is miR-21.

B
Build

To test the expression efficiency of the molecular switches we designed, while unifying our characterization criteria, data from different test batches can be compared. We followed the plasmid design in cycle3 while designing specific primers to modify the CAG repeat number in the inhibition sequence to 3× and 10×, CAG3 mRNA and CAG10 mRNA with corresponding switch sequences were obtained by in vitro transcription.

T
Test

The prepared CAG3 mRNA and CAG10 mRNA were compared with the CAG30 mRNA prepared in cycle1 and miR-21 by lipid nanoparticles (LNP) into 293T cell lines, and fluorescence signals were detected in the B610 channel by flow cytometry after 24h of culture.

![Figure 20: Fluorescence detection results of protein expression regulation by 30xCAG, 10xCAG, and 3xCAG sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle20.webp)

Figure 20. Fluorescence detection results of protein expression regulation by 30xCAG, 10xCAG, and 3xCAG sequences mCherry fluorescence was detected by flow cytometry and presented as Mean Fluorescence Intensity (MFI). The NC group was not transfected with any RNA. Statistical significance was assessed using ANOVA combined with Bonferroni's multiple comparison test (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

L
Learn

The experimental results showed that the control group transfected with CAG10 mRNA and the experimental group transfected with CAG10 mRNA and miR-21 mimic were detected by flow cytometry using B610 channels. There was no significant difference in mCherry fluorescence intensity (p>0.05). However, the control group transfected with CAG3 mRNA and the experimental group transfected with CAG3 mRNA and miR-21 mimic showed a significant difference in fluorescence intensity (p<0.01). Similarly, the control group transfected with CAG30 mRNA and the experimental group transfected with CAG30 mRNA and miR-21 mimic also showed a significant difference in fluorescence intensity (p<0.001). Based on the experimental results, we guessed that the better CAG repeat number might be around or above 30. As for the regulatory effect of CAG3 mRNA, it may be caused by other mechanisms, which needs to be further explored.

# Expression Vector Cycle

## Cycle 1

D
Design

We expect to give mRNA drug delivery vectors tumor-specific targeting capabilities through engineering design. Based on the literature review, we selected GPC3 protein, a member of the glycosaminoglycan family, which is highly expressed in 70–80% of patients with hepatocellular carcinoma, especially in poorly differentiated tumors, as a potential delivery recognition molecule , it is proposed to confer the specific recognition ability of HCC cells by conjugating GPC3 antibodies on the surface of lipid nanoparticles (LNPs) .
The specific strategy consists of three steps: (i) presentation of GPC3 antibodies using the Lpp-OmpA display system in E. coli BL21; (ii) Preparation of extracellular vesicles (EVS) that present GPC3 antibodies on the surface; (iii) It was heterozygated with LNPs to prepare LNP-EV heterozygous vectors carrying GPC3 antibodies on the surface.
As a confirmatory experiment, we first constructed an Lpp-OmpA-eGFP protein presentation system to present eGFP protein on the surface of Escherichia coli, and then prepared EVSThe protein expression level was also detected to verify the feasibility of the design idea.

B
Build

We used the pET-28a (+) plasmid as a vector to construct an expression vector for the presentation of eGFP on the surface of Escherichia coli in BL21. The plasmid contains a sequence encoding the Lpp-OmpA-eGFP fusion protein, which integrates lactose operons into the expression vector to regulate protein expression. At the same time, 6× His tags were added to the end of the fusion protein for Western Blot experimental characterization.

![Figure 21: eGFP Surface Display Expression Vector](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle21.webp)

Figure 21. eGFP Surface Display Expression Vector

T
Test

The plasmid was transformed into the BL21 strain. The bacteria were cultured in a 100 ml system until the OD600 reached 0.5. Protein expression was then induced by adding 50 μl of IPTG, followed by overnight incubation at 18°C. EVs were prepared by ultrasonic centrifugation after fragmentation of cells, and the fluorescence intensity of EVs was detected by nanoflow cytometry to characterize the number of eGFP surface presentations.

![Figure 22: Nanostream flow cytometry characterization of fluorescence intensity in eGFP-presenting EVs](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle22.webp)

Figure 22. Nanostream flow cytometry characterization of fluorescence intensity in eGFP-presenting EVs a: Test sample: eGFP-presenting EVs b: Test sample: EVs derived from wild-type BL21.

L
Learn

Through the nanoflow cytometry characterization results, we can indicate that the target protein has been presented on the surface of EVs, which basically verifies the feasibility of our Lpp-OmpA display system and can be used for the next characterization experiment.

## Cycle 2

D
Design

In cycle1, we basically validated the feasibility of our Lpp-OmpA demonstration system, and further we conjugated a single-stranded antibody of the GPC3 protein to the OmpA protein. The C-terminus of the EVS was prepared to surface display GPC3 single-stranded antibodies for subsequent heterozygous vector preparation.

B
Build

We used the pET-28a (+) plasmid as a vector to construct an expression vector for the presentation of GPC3 single-stranded antibodies on the surface of E. coli. The plasmid contains a sequence encoding the Lpp-OmpA-GPC3 single-stranded antibody fusion protein, integrating lactose operons into the expression vector to regulate protein expression, and adding 6xHis tags to the fusion protein end for Western blots experimental characterization.

![Figure 23: Surface display expression vector for GPC3 single-chain antibody](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle23.webp)

Figure 23. Surface display expression vector for GPC3 single-chain antibody

T
Test

The expression plasmid was transformed into BL21 strain. The bacteria were cultured in a 100 ml system until the OD 600 reached 0.5. Protein expression was then induced by adding 50 μl of IPTG, followed by overnight incubation at 18°C. EVS were prepared by ultrasonic centrifugation after crushing cells, and then Western blot assay was performed to detect the expression of target protein.

![Figure 24: Western blot results for EVs displaying eGFP and GPC3 monoclonal antibodies on their surfaces](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-engineering-cycle24.webp)

Figure 24. Western blot results for EVs displaying eGFP and GPC3 monoclonal antibodies on their surfaces. Lane 1 shows EVs produced by wild-type BL21 as a negative control. Lanes 2 and 3 display EVs presenting eGFP protein on their surfaces. Lane 4 shows EVs presenting GPC3 monoclonal antibodies on their surfaces. The expected molecular weights for both the Lpp-OmpA-eGFP fusion protein and the Lpp-OmpA-GPC3 monoclonal antibody fusion protein are approximately 42 kDa. The protein loading amount was 5 ng.

L
Learn

In this cycle, we performed Western Blot experimental characterization of EVs displaying eGFP and GPC3 single-stranded antibodies on the surface. From the development results, we successfully presented these two proteins on the surface of EV s, which further proved the effectiveness of our protein presentation system design and laid the foundation for our subsequent LNP-EVS hybridization experiments.

# References

* HUANG A, YANG X-R, CHUNG W-Y, et al. Targeted therapy for hepatocellular carcinoma [J]. Signal Transduction and Targeted Therapy, 2020, 5(1): 146.
* AN S, FU L. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs [J]. EBioMedicine, 2018, 36: 553-62.
* XUE X, ZHANG C, LI X, et al. mRNA PROTACs: engineering PROTACs for high-efficiency targeted protein degradation [J]. 2024, 5(2): e478.
* VOGEL A, MEYER T, SAPISOCHIN G, et al. Hepatocellular carcinoma [J]. The Lancet, 2022, 400(10360): 1345-62.
* Lv, DD., Zhou, LY. & Tang, H. Hepatocyte nuclear factor 4α and cancer-related cell signaling pathways: a promising insight into cancer treatment. Exp Mol Med 53, 8–18 (2021). https://doi.org/10.1038/s12276-020-00551-1
* YANG T, POENISCH M, KHANAL R, et al. Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model [J]. Journal of Hepatology, 2021, 75(6): 1420-33.
* Wu, SH., Xiao, MC., Liu, F. et al. Cell-permeated peptide P-T3H2 inhibits malignancy on hepatocellular carcinoma through stabilizing HNF4α protein. Discov Onc 15, 752 (2024). https://doi.org/10.1007/s12672-024-01661-2
* MitchellHo, HeungnamKim, Glypican-3: A new target for cancer immunotherapy, European Journal of Cancer. Volume47, Issue 3, 2011, Pages 333-338, ISSN 0959-8049.
* BELAIR D G, LEE J S, KELLNER A V, et al. Receptor mimicking TGF-β1 binding peptide for targeting TGF-β1 signaling [J]. Biomaterials Science, 2021, 9(3): 645-52.
* PAN Y, LU J, FENG X, et al. Gelation of cytoplasmic expanded CAG RNA repeats suppresses global protein synthesis [J]. Nature Chemical Biology, 2023, 19(11): 1372-83.
* FUJITA Y, HIROSAWA M, HAYASHI K, et al. A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches [J]. Science Advances, 8(1): eabj1793.
* SHI H, WEN SU W. Display of green fluorescent protein on Escherichia coli cell surface [J]. Enzyme and microbial technology, 2001, 28(1): 25-34.
* ALTER C L, LOTTER C, PULIGILLA R D, et al. Nano Plasma Membrane Vesicle-Lipid Nanoparticle Hybrids for Enhanced Gene Delivery and Expression [J]. 2025, 14(1): 2401888.
* WANG S, EMERY N J, LIU A P. A Novel Synthetic Toehold Switch for MicroRNA Detection in Mammalian Cells [J]. ACS Synthetic Biology, 2019, 8(5): 1079-88.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}

![iGEM lead-img](https://static.igem.wiki/teams/5844/lead/dut-china-experiment.webp)

{% endblock %}
{% block page\_content %}

[Overview](#overview)
[RNA Transcription](#rna-transcription)
[mRNA Switch Transfection](#mrna-switch-transfection)
[5'-UTR Validation](#mrna-utr-validation)
[Western Blot](#western-blot)
[Cell Passaging](#cell-passaging)
[qPCR](#qpcr)
[siRNA Transfection](#sirna-transfection)
[Delivery Vectors](#delivery-vectors)

## Overview

This experiment section systematically presents the key experimental procedures and technical validations conducted by the iGEM DUT-China team. Starting with in vitro transcription and modification of RNA, we verified the switch mechanism and 5'-UTR functionality through mRNA cell transfection. We quantitatively analyzed expression outcomes at both protein and RNA levels using techniques such as Western Blot and qPCR. Additionally, we explored siRNA transfection conditions and developed a hybrid delivery system based on extracellular vesicles (EVs) and lipid nanoparticles (LNPs), which was thoroughly characterized. All experiments were designed rigorously with clear protocols, providing reliable data to support the functional validation of synthetic biological components and the optimization of delivery systems.

## 1. RNA in vitro transcription experiment

### Step1. PCR amplification of plasmid template

**Required reagents**

* 2X PCR Mix Purple Enzyme: High-fidelity DNA polymerase, dNTPs (deoxyribonucleoside triphosphates), buffer mixture
* Plasmid template (50 ng/μL)
* Designed and ordered primers (10 μM)

**Steps:**

1. Calculate the system concentration to be configured in advance and take out the reagents and thaw them on ice in advance;

2. Thaw high-concentration (>36 ng/μL) and dry powder systems by flicking to mix thoroughly. Centrifuge briefly to remove primers and other components to the bottom of the tube.

3. Dilute the plasmid to 5 ng/μL (prepared with 100 μL) with sterile water;

4. Dilute the primer freeze-dried powder with sterile water to prepare a 10 μM (μmol/L) primer solution, then vortex to mix thoroughly.

5. Preheat the PCR instrument cover in advance

6. Add the following volumes of each component into a PCR tube (the smallest centrifuge tube) to prepare the PCR reaction solution (amplify 50 μL of system sample, prepare the sample on ice ).

Prepare PCR reaction system (50 μL) on ice

* T plasmid DNA template (10 ng) 2 μL
* Primer Forward, F forward (10μM) 2 μL
* Primer Reserve, R reaction material (10μM) 2 μL
* 2× BeyoFusion ™ PCR Master Mix Purple 25 μL
* ddH2O enzyme-free water Make up to 50 μL (19 μL)

\*Note: The order of adding samples is ddH2O, F, R, T, Mix (PCR Mix enzyme must be added last)

7. After adding the components, carefully mix the PCR reaction solution by flicking it with your fingers or turning it upside down. Centrifuge briefly to allow the PCR reaction solution to accumulate at the bottom of the tube. Keep the PCR reaction solution on ice if it is not used immediately.

8. Place the configured PCR reaction system on the PCR instrument, set the program and start the PCR reaction:

PCR instrument reaction parameter setting

* 1) Pre-denaturation 98℃ 30 s
* 2) transsexual 98℃ 10 s
* 3) annealing Annealing temperature\* 5 s
* 4) extend 72℃ 5s/kb \*\*
* Steps 1 to 4 , cycle 30 times
* 5) Final extension 72℃ 1 min

\* The annealing temperature is set based on the contact part of the primers, generally between 52-61°C, 3-5°C below Tm.

Use a gradient method: pre-run the experiment first, then perform large-scale PCR. Divide the sample from one PCR tube into five tubes. Each column of samples in the PCR machine is set to the same temperature, while different columns are set to different temperatures. The optimal temperature is determined simultaneously.

The extension time is 5 seconds per kb, and the time can be extended appropriately based on this. The extension time is based on the longest sequence in the sample (the length of the sequence between the two primers to be P, including the plasmid). For example, if the sequence length is 4400 bp, 4.4\*5=22 seconds. Based on this, the extension time can be extended by a few seconds, such as 25 seconds.

9. Load the sample and start the reaction. While waiting for the reaction, prepare the gel. After the reaction is complete, place the amplified product on a PCR tube rack for electrophoresis.

### Step2. Agarose electrophoresis verification

**Required reagents:**

* 1% agarose gel
* DNA Marker

**Steps:**

1. Preparation of gel: Weigh 0.4g of agarose using weighing paper and add it to a beaker. Then add 40ml of TAE solvent to the beaker, seal it with plastic wrap and a rubber band, and heat it in a microwave for a while. Remove the beaker and check if there are any small solid particles in the liquid. Heat it again until the agarose is completely dissolved and the liquid becomes transparent. After the liquid cools, add 4μL of nucleic acid dye and shake well.

2. Pour into the gel plate and wait for it to dry thoroughly (>30min);

3. Load the sample into the gel wells. Place one PCR product of each sequence (6 μL system, 1× buffer 5 μL + product 1 μL, or 6× buffer 1 μL + product 1 μL + enzyme-free water 4 μL) in one well, and place 5x DNA Marker (5 μL) in the next well.

4. Move the gel to the leftmost side of the container and align it. Run the gel at 180V for 22 minutes.

5. Use UV imager to observe whether the band size is consistent with the expected

6. Select the PCR product with the brightest color and lightest shadow corresponding to the temperature of the band and proceed to the next step

\*Note: The results here can be stored for one day

**Troubleshooting:**

* No band: Check primer design/annealing temperature
* Non-specific bands: Optimize annealing temperature or redesign primers

### Step3. Kinase Domain Linker (KDL) connection

**Required reagents:**

* NEB KLD Enzyme Mix (10x)
* KLD Reaction Buffer (2x)

**Steps:**

1. First add 1 μL of PCR product, then add 1.25 μL of buffer, and finally add 0.25 μL of KLD enzyme;

Prepare the reaction system on ice (2.5 μL)

* PCR products 1 μL
* 2× KLD Reaction Buffer 1.25 μL
* 10× KLD enzyme  0.25 μL

\*Note: The system can be expanded by 4 or 8 times as needed

2. Mix gently by flicking and react at room temperature for 10 min.

### Step4. E. coli transformation

**Required reagents:**

* DH5α competent cells (thawed on ice)
* SOC medium (preheated to 37°C) in a 4°C refrigerator
* LB plate (Kan antibody)

**Steps:**

1. Open a water bath (37°C, 42°C) in advance and thaw the competent cells on ice for 2-3 minutes (semi-solid, semi-liquid is sufficient).

2. Light the alcohol lamp and place 100 μL of competent cells in a 1.5 ml centrifuge tube (be careful not to shake the cells vigorously).

3. Add 2.5 μL of the KLD-ligated product to a 1.5 ml centrifuge tube and mix gently.

4. Place in an ice bath for 30 minutes, then quickly heat shock in a 42°C water bath for 45 seconds, then quickly transfer to ice and let stand for 2 minutes.

5. Light an alcohol burner and add 900 μL of SOC medium (pre-warmed at 37°C) to a 1.5 ml centrifuge tube.

6. Resuscitate at 37°C for 1 hour;

7. Concentration: Centrifuge at 5000 x g for 1 min (cells will settle at the bottom of the tube after centrifugation ). Aspirate and discard 850 μL of supernatant. Mix the remaining 100 μL by pipetting ( re-aspirate the liquid with a pipette and gently pipette to redisperse the cells . Avoid violent shaking to prevent cell damage). Ensure that the pellet is completely dispersed.

8. Spread 100 μL of bacterial solution on a 100 mg/L Kan resistance (same resistance as plasmid selection ) LB plate and incubate at 37°C until the bacterial solution is absorbed.

9. Place the plate upside down on a 37°C shaker at 10 rpm, press and hold the start button, and incubate for 12-16 hours (the specific time depends on the size of the colony);

If the time is not right, you can wrap the plate with sealing film and store it in a 4℃ refrigerator, and then pick out single bacteria later.

10. The next day, pick 3-4 single colonies from different areas of the plate and place them in 3-4 bottles. Add 4ml of liquid culture medium to each bottle. Heat the bottle neck and cap with an alcohol burner before capping. Do not cap the bottle neck too tightly, leaving enough air for bacterial growth. Incubate the plate in a 37°C shaker with full agitation for 12-16 hours (<20 hours). After use, wrap the plate with sealing film and store it upside down in a 4°C refrigerator.

### Step5. Plasmid amplification

**Required reagents:**

* LB liquid medium (Kana resistance, self-made)

**Steps:**

1. Place 10-20 ml of LB medium (Kana resistance) into a sterilized 50 ml centrifuge tube.

2. Place the glycerol bacteria cryovial on ice and add 10-20 μL of the thawed glycerol bacteria to 5 ml of LB medium.

3. Place the test tube in an incubator at 37°C and 200 rpm for overnight culture (12-16 h).

### Step6. Plasmid extraction

**Required reagents:**

* FastPure EndoFree Plasmid Mini Plus Kit

\*Note: Buffer P2 is prone to precipitation at low temperatures (≤20°C). When the room temperature is ≤20°C, it needs to be heated at 37°C for 10 minutes to completely dissolve. Cool to room temperature and mix thoroughly before use.

**Steps:**

\*Note: Preheat enzyme-free water to 65°C in a metal bath in advance.

1. Take 5-15 ml of overnight culture and centrifuge at 12,000 rpm (13,400 × g) for 1 min to collect the bacteria. Aspirate as much of the remaining supernatant as possible (discard the supernatant).

2. Add 500 μl of Buffer P1 (stored in the second layer of a 4°C refrigerator. Please check whether RNase A Solution has been added first). Vortex until the cells are completely resuspended. Transfer the resuspended liquid to a 2 ml centrifuge tube.

3. Immediately add 500 μl of Buffer P2, mix gently by inverting up and down 8-10 times, and let it stand at room temperature for 3 minutes (timer).

\*Note: This step should be mixed gently and do not shake violently to avoid genomic DNA contamination.

At this point, the solution should gradually change from a turbid, viscous blue to a clear, viscous blue, indicating complete lysis. The time should not exceed 5 minutes to avoid plasmid damage. When working with multiple samples simultaneously, mix each tube thoroughly and then time the reaction.

If the solution does not become clear, it may be due to excessive bacterial cells and incomplete lysis. The amount of bacterial cells should be reduced.

4. Add 500 μl of Buffer N3 and immediately and gently invert the tube 12-15 times until the blue color disappears completely and a white flocculent precipitate appears. Incubate at room temperature for 10 min and centrifuge at 12,000 rpm (13,400 × g) for 10 min.

\*Note: Immediately after adding Buffer N3, gently invert the tube to mix thoroughly to avoid local precipitation. At this point, the solution will turn from blue to colorless, indicating complete neutralization. After centrifugation, the supernatant should be clear. If a small white precipitate appears on the surface of the supernatant, it will not affect subsequent operations. Wait 10 minutes before proceeding to the next step of column equilibration.

5. Column equilibration: Place the FastPure DNA Mini Columns III column in a 2 ml Collection Tube. Add 200 μl of Buffer QB to the FastPure DNA Mini Columns III column and centrifuge at 12,000 rpm (13,400 × g) for 1 min. Discard the filtrate and set aside.

\*Note: The equilibration solution can activate the silica membrane. Please use the column that has been processed on the same day.

6. Transfer the supernatant (about 1.5 ml) to a 5 ml centrifuge tube (self-prepared), add 0.3 times the volume of supernatant (about 450 μl) of isopropanol, and mix thoroughly by inverting the tube 10-15 times.

\*Note: Adding too much isopropanol can easily lead to RNA residue. Please add isopropanol according to the actual volume of the supernatant.

If you cannot aspirate all the supernatant, add a small amount of isopropanol, such as 435 or 440 μl.

7. Transfer the mixed solution from step 6 to the adsorption column, centrifuge at 12,000 rpm (13,400 × g) for 1 min, and discard the filtrate.

\*Note: Allow to stand for 1 minute before centrifugation. The maximum volume of the adsorption column is 750 μl. If the mixture exceeds this volume, apply it to the column in multiple batches, adding a maximum of 700 μl each time.

8. Repeat step 7 until all the mixed solution is loaded onto the column.

9. Add 500 μl of Buffer PW1 to the adsorption column, centrifuge at 12,000 rpm (13,400 × g) for 1 min, and discard the filtrate.

10. Add 600 μl of Buffer PW2 (please check whether anhydrous ethanol has been added) to the adsorption column, centrifuge at 12,000 rpm (13,400 × g) for 1 min, and discard the filtrate.

11. Repeat step 10.

12. Place the adsorption column back into the collection tube and dry it by centrifugation at 12,000 rpm (13,400 × g) for 2 min.

\*Note: After this step, you can open the lid and let it sit at room temperature for 3-5 minutes.

13. Place the column in a new 1.5 ml microcentrifuge tube and add 60–200 μl (100 μl) of 65°C preheated Endotoxin-free Elution Buffer (with ddH2O for subsequent sequencing or enzyme digestion) to the center of the column membrane. Incubate at room temperature for 1 minute. Centrifuge at 12,000 rpm (13,400 × g) for 1 minute and discard the column.

\*Note: The recommended elution volume is no less than 60 μl. A smaller volume will reduce the elution efficiency.

\*Note: If ddH2O is used for elution, the pH of ddH2O should be within the range of 7.0 - 8.5. A pH below 7.0 will reduce the elution efficiency.

\*Note: To increase the elution efficiency of the plasmid, the elution buffer can be preheated at 65°C. To increase the elution yield, the centrifuge buffer can be loaded onto the column for a second elution.

14. Measure the concentration of the extracted plasmid. Wash the upper and lower wells twice with enzyme-free water, wipe dry the upper and lower wells, apply nucleic acid detection, apply DNA, use the liquid for eluting DNA (here is enzyme-free water in a metal bath at 65°C), apply blank calibration, wipe dry, and add the eluted DNA liquid.

15. The obtained plasmid DNA solution was stored at -20°C or used for subsequent experiments.

### Step7. DNA template linearization by single enzyme digestion

**Required reagents:**

* Restriction enzyme BspQI endonuclease (selected according to the multiple cloning site, placed on the left side of the -20°C refrigerator);
* gel red
* Marker

**Steps:**

1. Open a 50℃ water bath in advance.

2. Prepare the enzyme digestion system as follows (on ice):

Enzyme digestion system (120 μL)

* plasmid DNA 100 μL
* 10x Buffer r3.1 12 μL
* BspQI (10U/μL)\* 2 μL
* ddH2O 6 μL to make up to 120 μL

\*Mix the buffer, template, and water thoroughly before adding the endonuclease. Keep the endonuclease on ice when using .

\*The enzyme digestion system can be flexibly scaled according to the concentration of plasmid DNA

3. Incubate in a 50°C water bath for 2 hours or longer;

4. 1% agarose gel electrophoresis to check whether the enzyme digestion is complete and the length is consistent with the expected. Sample system (6 μL)

Add 5ul to one 5× Marker well , one plasmid well (1× buffer 5μL + plasmid product 1ul, or 6× buffer 1μL + plasmid product 1ul + enzyme-free water 4ul), and one enzyme-digested product well (1× buffer 5μL + enzyme-digested product 1ul, or 6× buffer 1μL + enzyme-digested product 1ul + enzyme-free water 4ul).

### Step8. Purification of enzyme digestion products

**Required reagents:**

* Column-type PCR product purification kit (Shengong column-type)

**Steps:**

1. Prepare the enzyme digestion system as follows: Transfer the digestion reaction solution (centrifuge to remove water vapor) to a clean 1.5 mL centrifuge tube, add 5 times the volume of Buffer B3 (for binding), and mix thoroughly.

2. Transfer all the mixed liquid into the adsorption column and let it stand for a while to allow it to bind better. Centrifuge at 8000×g for 30 s and time for 1 min. Pour out the liquid in the collection tube and place the adsorption column into the same collection tube.

3. Add 500 μL of Wash Solution (rinsing) to the adsorption column, centrifuge at 9000 × g for 30 s and time 1 min, and discard the liquid in the collection tube.

4. Repeat step 3 once .

5. Leave the container open at room temperature for 10 min (to completely evaporate the ethanol).

6. The empty adsorption column and collection tube were centrifuged at 9000 × g for 1 min.

7. Place the adsorption column in a clean 1.5 mL centrifuge tube and add 15–40 μL (depending on subsequent requirements) of enzyme-free Elution Buffer (preheated at 65°C) to the center of the adsorption membrane. Note: To account for evaporation losses, do not add the exact amount, but add a little more. Incubate at 65°C for 1–2 minutes, then centrifuge at 9000 × g for 1 minute. Store the resulting plasmid DNA solution at -20°C or use it in subsequent experiments.

8. The concentration of the obtained DNA solution was measured on an ultramicro spectrophotometer.

### Step9. In vitro transcription and synthesis of mRNA

**Required reagents:**

* High Yield T7 RNA Synthesis Kit
* Cap analogs
* N1-Me-Pseudo UTP

**Steps:**

1. Prepare the reaction system in the following order (room temperature):

In vitro transcription system (20 μL)

* Nuclease-free Water 20 μL
* 100mM ATP Solution 1.6 μL
* 100mM GTP Solution 1.6 μL
* 100mM CTP solution 1.6 μL
* 100mM N1-Me-Pseudo UTP solution 1.6 μL
* 100mM Cap Analog 1.6 μL
* Template DNA X μL (1 μg)
* 5x/10x Reaction Buffer 4 μL/2 μL
* Enzyme Mix 1.5 μL

2. Gently invert the reaction system to mix well, and centrifuge briefly to allow the liquid to accumulate at the bottom;

3. Incubate at 37°C for 2 hours;

4. Add 1 U of DNase I, mix well, and incubate in a 37°C metal bath for 15 min.

### Step10. Purification of transcribed mRNA products

**Required reagents:**

* SteadyPure Universal RNA Extraction Kit II (AG)

**Steps:**

1. Preparation steps: Preheat (100\*n + appropriate amount) μL of enzyme-free water to 65°C. (If you have several tubes of product, preheat a few hundred μL, and preheat more to allow for evaporation losses)

2. Add enzyme-free water to the sample to make up to 100 μL and mix gently but thoroughly.

3. Add 400 μL of Buffer RLS to the sample and mix gently using a pipette .

4. Add 250 μL of absolute ethanol to the sample and mix gently using a pipette .

5. Transfer the sample to Universal RNA Mini Columns, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

6. Add 600 μL of Buffer RWA, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

7. Add 650 μL of Buffer RWB, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

8. Add 650 μL of Buffer RWB, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

9. The adsorption column was placed in a new collection tube and centrifuged at 12,000 rpm for 1 min.

10. Place the adsorption column in a new enzyme-free centrifuge tube, add 100 μL of enzyme-free water preheated at 65°C to the center of the column membrane , cover the tube, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, and collect the eluate.

11. Measure the concentration of mRNA product.

## 2. mRNA cell transfection experiment for switch verification

### Step1. LNP solution preparation

**Required reagents:**

LNP component of 1273 formulation

formula:

LNP components of formula 1273 , molar ratio: DOTAP: cholesterol :DSPC:PEG- lipid = 50:38.5:10:1.5

Liposome concentration ratio ( Formula 1273 )

| Components | content | Mv | Weigh the mass | concentration | Weigh the volume |
| --- | --- | --- | --- | --- | --- |
| HUO | 50% | 709 | 0.008863 g | 0.1 g/mL | 88.63μL |
| D0PE | 10% | 744.034 | 0.00186 g | 0.1 g/mL | 18.60μL |
| cholesterol | 37.5% | 386.65 | 0.003635 g | 0.02 g/mL | 181.75μL |
| DSPEPEG2000 | 2.5% | 2000 | 0.001250 g | 0.01 g/mL | 125μL |

**Steps:**

1. Calculate the required LNP volume based on the RNA quantity. For 23 mRNA sequences, each requiring 100 μg of mRNA, the total mRNA mass required is 23 \* 100 = 2300 μg, or 2300,000 ng. The concentration of each mRNA is 170 ng/ μL . Therefore, the total mRNA volume required is 2300,000 ng ÷170 ng/ μL = 13529.4118 μL . Since the mRNA:LNP volume ratio is 3:1, the required LNP volume is 13529.4118 μL ÷ 3 = 4509.8039 μL , or 5 ml.

2. First, transfer 5ml of anhydrous ethanol to a centrifuge tube. HUO (SM-102) is a viscous liquid, so add 1ml of anhydrous ethanol to the HUO (SM-102) centrifuge tube. Then, use a precision balance to weigh each component and record the actual weight .

3. Liposome preparation (concentration of 12.5 mM): Use a micropipette to accurately weigh the corresponding volumes of cationic carrier, amphiphilic phospholipid bimolecule , cholesterol, and DSPE-PEG2000 into a 2 mL centrifuge tube, and add 100% ethanol to make up the system to 2 mL.

4. The fully dissolved liquid was dispensed into small centrifuge tubes, sealed with sealing strips and stored in a 4°C refrigerator.

### Step2. Reagent preparation

1. DMEM complete medium: 90 mL DMEM basal medium + 10 mL special fetal bovine serum + 1 mL penicillin-streptomycin solution (100×), i.e. 10% serum + 1% double antibody;

2. Serum-free DMEM medium: 100 mL DMEM basal medium + 1 mL penicillin-streptomycin solution (100×);

3. Maintenance medium: serum-free medium + DMEM complete medium 1:1 mixture, i.e. 5% serum + 1% double antibody;

4. Trypsin cell digestion solution (0.5×): 10 mL trypsin cell digestion solution + 10 mL PBS (1×). The storage period is 1 month at 4°C. Prepare the appropriate amount according to the usage. 20 mL can be used about 15 times.

5. Other reagents:

| Name | Brand | Storage conditions |
| --- | --- | --- |
| PBS buffer (1×) | Corning | 4℃ |
| Citrate buffer (pH 4.0, 50 mM) | Self-configured | 4℃ |
| LNPs | Self-configured | 4℃ |
| Firefly luciferase detection reagent | Yisheng | -20℃ |

### Step3. Cell seeding

1. When the confluence of cells is 70-80%, discard the original culture medium and add appropriate amount of PBS (1×) to wash 2-3 times.

2. Add an appropriate amount of trypsin digestion solution (0.5×) (just enough to cover the bottom of the bottle), place the tube in a 37°C 5% CO2 incubator and incubate for 1 min. Observe under a microscope until the cells are about to become round. Immediately discard the trypsin and add 2-3 mL of DMEM complete medium. Pipette the cells off the bottle wall and mix thoroughly.

3. Transfer the cell suspension to a clean 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add about 5 mL of new DMEM complete medium, and mix thoroughly by pipetting.

4. Take about 10 μL of cell suspension and add it to the cell counting plate. Insert the side with the cell suspension inward into the cell counter, select the automatic mode, and read the concentration of the cell suspension.

5. After counting, the cell suspension was diluted to a concentration of 80,000-100,000 cells/mL.

6. Add 100 μL of cell suspension to each well of a 96-well plate (i.e., 8,000-10,000 cells per well).

\*After adding each set of control wells, the cell suspension should be remixed to ensure that the difference in cell concentration is not large;

\*PBS was added to the outermost circle of culture wells in the 96-well plate and evaporated;

\*Do not let the gun tip touch the centrifuge tube mouth or other places. If it touches, replace the gun tip immediately.

7. Place in a 37°C 5% CO2 incubator and culture for 18-24 h.

### Step4. Cell transfection

1. When the cell confluence in the plate reaches about 80%, transfection is performed.

2. Dilute the Toehold mRNA to be transfected to 170 ng/μL with citrate buffer (pH 4.0, 50 mM).

3. After mixing mRNA, miRNA, and miRNA inhibitors, mix mRNA:LNPs , miRNA inhibitors, or miRNA:LNPs at a ratio of 3:1 (6 μL mRNA + 3 μL miRNA + 3 μL LNPs) by pipetting and incubate at room temperature for 10 min.

4. While waiting for the incubation of mRNA and liposomes, process the cells: discard the original culture medium in the 96-well plate, add an appropriate amount of PBS (1×) to wash once, add 50 μL of DMEM complete culture medium to each well, and place in the incubator for 5 minutes.

5. Mix the "mRNA-miRNA-LNPs" mixed solution again, and add 49 μL of serum-free DMEM medium to every 1 μL of mRNA-miRNA-LNPs mixed solution (for example, add 392 μL of serum-free DMEM medium to 8 μL of mRNA-miRNA-LNPs mixed solution).

6. Add 50 μL of the "mRNA-miRNA-LNPs" mixed solution (added by adhering to the wall) to each well, shake gently to mix, and place in a cell culture incubator.

### Step5. Fluorescence detection

When used for the first time, pour all the Firefly Luciferase Buffer into the Firefly Luciferase Substrate Bottle at once, mix thoroughly, and then divide according to the usage requirements. It is recommended to store it at -70℃ for a long time or at -20℃ for no more than one month. After the test reagents are divided and frozen, they need to be equilibrated to room temperature before each subsequent experiment. Note: The Firefly Luciferase Buffer can be thawed at room temperature or in a water bath, but the temperature should not exceed 25℃.

**Operation steps**

1. Remove the culture plate containing mammalian cells from the cell culture incubator and allow to equilibrate to room temperature for 5-15 minutes.

2. Add detection reagent: Add an equal volume of detection reagent to the test cell culture medium and equilibrate to room temperature (for example, a 96-well plate usually adds 100 μl of culture medium and correspondingly adds 100 μl of detection reagent; a 384-well plate usually adds 30 μl of culture medium and correspondingly adds 30 μl of detection reagent).

3. Oscillating and mixing: To ensure sufficient cell lysis, it is recommended to mix on a horizontal shaker at room temperature for 5-10 minutes (Note: Do not use a pipette to mix, as bubbles will affect the luminescence detection reading. The mixing time can be adjusted appropriately according to the cell number to ensure sufficient cell lysis and obtain stable luminescence detection results).

4. Detection: Detect the luminescent signal on a chemiluminescence detector or a multifunctional microplate reader with a chemiluminescence module. Complete the detection within 2 hours after adding the detection reagent.

## 3. mRNA Cell Transfection Experiment for 5- UTR Validation

### Step1. LNP solution preparation

**Required reagents:**

LNP component of 1273 formulation

formula:

LNP components of formula 1273 , molar ratio: DOTAP: cholesterol :DSPC:PEG- lipid = 50:38.5:10:1.5

Liposome concentration ratio ( Formula 1273 )

| Components | content | Mv | Weigh the mass | concentration | Weigh the volume |
| --- | --- | --- | --- | --- | --- |
| HUO | 50% | 709 | 0.008863 g | 0.1 g/mL | 88.63μL |
| D0PE | 10% | 744.034 | 0.00186 g | 0.1 g/mL | 18.60μL |
| cholesterol | 37.5% | 386.65 | 0.003635 g | 0.02 g/mL | 181.75μL |
| DSPEPEG2000 | 2.5% | 2000 | 0.001250 g | 0.01 g/mL | 125μL |

**Steps:**

1. Calculate the required LNP volume based on the RNA quantity. For 23 mRNA sequences, each requiring 100 μg of mRNA, the total mRNA mass required is 23 \* 100 = 2300 μg, or 2300,000 ng. The concentration of each mRNA is 170 ng/ μL . Therefore, the total mRNA volume required is 2300,000 ng ÷170 ng/ μL = 13529.4118 μL . Since the mRNA:LNP volume ratio is 3:1, the required LNP volume is 13529.4118 μL ÷3 = 4509.8039 μL , or 5 ml.

2. First, transfer 5ml of anhydrous ethanol to a centrifuge tube. HUO (SM-102) is a viscous liquid, so add 1ml of anhydrous ethanol to the HUO (SM-102) centrifuge tube. Then, use a precision balance to weigh each component and record the actual weight .

3. Liposome preparation (concentration of 12.5 mM): Use a micropipette to accurately weigh the corresponding volumes of cationic carrier, amphiphilic phospholipid bimolecule , cholesterol, and DSPE-PEG2000 into a 2 mL centrifuge tube, and add 100% ethanol to make up the system to 2 mL.

4. The fully dissolved liquid was dispensed into small centrifuge tubes, sealed with sealing strips and stored in a 4°C refrigerator.

### Step2. Reagent preparation

1. DMEM complete medium: 90 mL DMEM basal medium + 10 mL special fetal bovine serum + 1 mL penicillin-streptomycin solution (100×), i.e. 10% serum + 1% double antibody;

2. Serum-free DMEM medium: 100 mL DMEM basal medium + 1 mL penicillin-streptomycin solution (100×);

3. Maintenance medium: serum-free medium + DMEM complete medium 1:1 mixture, i.e. 5% serum + 1% double antibody;

4. Trypsin cell digestion solution (0.5×): 10 mL trypsin cell digestion solution + 10 mL PBS (1×). The storage period is 1 month at 4°C. Prepare the appropriate amount according to the usage. 20 mL can be used about 15 times.

5. Other reagents:

| Name | Brand | Storage conditions |
| --- | --- | --- |
| PBS buffer (1×) | Corning | 4℃ |
| Citrate buffer (pH 4.0, 50 mM) | Self-configured | 4℃ |
| LNPs | Self-configured | 4℃ |
| Firefly luciferase detection reagent | Yisheng | -20℃ |

### Step3. Cell seeding

1. When the confluence of cells is 70-80%, discard the original culture medium and add appropriate amount of PBS (1×) to wash 2-3 times.

2. Add an appropriate amount of trypsin digestion solution (0.5×) (just enough to cover the bottom of the bottle), place the tube in a 37°C 5% CO2 incubator and incubate for 1 min. Observe under a microscope until the cells are about to become round. Immediately discard the trypsin and add 2-3 mL of DMEM complete medium. Pipette the cells off the bottle wall and mix thoroughly.

3. Transfer the cell suspension to a clean 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add about 5 mL of new DMEM complete medium, and mix thoroughly by pipetting.

4. Take about 10 μL of cell suspension and add it to the cell counting plate. Insert the side with the cell suspension inward into the cell counter, select the automatic mode, and read the concentration of the cell suspension.

5. After counting, the cell suspension was diluted to a concentration of 80,000-100,000 cells/mL.

6. Add 100 μL of cell suspension to each well of a 96-well plate (i.e., 8,000-10,000 cells per well).

\*After adding each set of control wells, the cell suspension should be remixed to ensure that the difference in cell concentration is not large;

\*PBS was added to the outermost circle of culture wells in the 96-well plate and evaporated;

\*Do not let the gun tip touch the centrifuge tube mouth or other places. If it touches, replace the gun tip immediately.

7. Place in a 37°C 5% CO2 incubator and culture for 18-24 h.

### Step4. Cell transfection

1. Transfection can be performed only when the cell confluence reaches about 80%.

2. Dilute the mRNA to be transfected to 170 ng/μL with citrate buffer (pH 4.0, 50 mM).

3. Mix mRNA:LNPs at a ratio of 3:1 by pipetting (customary method: 6 μL mRNA + 2 μL LNPs mix) and incubate at room temperature for 10 min.

4. While waiting for the incubation of mRNA and liposomes, process the cells: discard the original culture medium in the 96-well plate, add an appropriate amount of PBS (1×) to wash once, add 50 μL of DMEM complete culture medium to each well, and place in the incubator for 5 minutes.

5. Mix the mRNA-LNPs mixed solution again, and add 49 μL of serum-free DMEM medium to every 1 μL of mRNA-LNPs mixed solution (for example, add 392 μL of serum-free DMEM medium to 8 μL of mRNA-LNPs mixed solution).

6. Add 50 μL of the "mRNA-LNPs-serum-free" mixed solution (added by adhering to the wall) to each well, vortex gently to mix, and place in a cell culture incubator.

### Step5. Fluorescence detection

When used for the first time, pour all the Firefly Luciferase Buffer into the Firefly Luciferase Substrate Bottle at once, mix thoroughly, and then divide according to the usage requirements. It is recommended to store it at -70℃ for a long time or at -20℃ for no more than one month. After the test reagents are divided and frozen, they need to be equilibrated to room temperature before each subsequent experiment. Note: The Firefly Luciferase Buffer can be thawed at room temperature or in a water bath, but the temperature should not exceed 25℃.

**Operation steps:**

1. Remove the culture plate containing mammalian cells from the cell culture incubator and allow to equilibrate to room temperature for 5-15 minutes.

2. Add detection reagent: Add an equal volume of detection reagent to the test cell culture medium and equilibrate to room temperature (for example, a 96-well plate usually adds 100 μl of culture medium and correspondingly adds 100 μl of detection reagent; a 384-well plate usually adds 30 μl of culture medium and correspondingly adds 30 μl of detection reagent).

3. Oscillating and mixing: To ensure sufficient cell lysis, it is recommended to mix on a horizontal shaker at room temperature for 5-10 minutes (Note: Do not use a pipette to mix, as bubbles will affect the luminescence detection reading. The mixing time can be adjusted appropriately according to the cell number to ensure sufficient cell lysis and obtain stable luminescence detection results).

4. Detection: Detect the luminescent signal on a chemiluminescence detector or a multifunctional microplate reader with a chemiluminescence module. Complete the detection within 2 hours after adding the detection reagent.

## 4. Western Blot

### Step1. Cell Lysis

After washing with 1 mL of PBS, each well was added with 500 μL of trypsin for 2 min. After digestion was complete, 800 μL of DMEM medium was added to completely terminate the digestion.

The cells were blown off the 6-well plate, harvested into a 1.5 mL centrifuge tube, and centrifuged at 1000× g for 1 min. The cell pellet was resuspended in PBS and centrifuged at 1000× g for 1 min at 4°C.

Resuspend with lysis buffer and lyse on ice for 30 minutes. Centrifuge the lysate at 1200xg for 10 minutes at 4°C, and carefully aspirate the supernatant into a new 1.5 mL centrifuge tube.

### Step2. Protein Concentration Determination

Prepare BCA standard (10ul BCA standard + 90ul PBS) and working solution ( BCA:Cu reagent = 50:1)

| Standard (ul) | 0 | 2 | 4 | 6 | 8 | 12 | 16 | 20 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PBS( ul) | 20 | 18 | 16 | 14 | 12 | 8 | 4 | 0 |

2ul sample + 18ul PBS (repeated 3 times)

Add 200ul of working solution to each well

Incubate at 37°C for 30 minutes.

Take out the sample and place it in the microplate reader. After measurement, use the standard to draw a standard curve.

The protein concentration of the samples was read using the standard curve.

The sample was added with 1/4 volume of 5× loading buffer and placed in a 100°C metal bath for 5 min, and then stored in a 4°C freezer.

### Step3. Protein Electrophoresis

Prepare electrophoresis gel according to the instructions.

Ensure that the sample volume and protein amount are consistent.

First use 80V electrophoresis to run the stacking gel, and then use 120~140V electrophoresis to run the separation gel.

Take out the gel after electrophoresis, cut off the irrelevant part, measure the size and cut the appropriate membrane, soak it in methanol and membrane activation solution for 1 minute respectively, and then place the electrophoresis gel and membrane on the transfer instrument for transfer.

After the transfer, seal the membrane with 5% skim milk powder in TBSD and place it at 60 rpm for 1 hour.

### Step4. Incubation with Antibodies

Prepare the primary and secondary antibodies according to the instructions . After sealing , add the primary antibody and incubate at 4°C overnight or at 100 rpm for 3 hours.

After incubation, wash three times with TBSD, each time for 10 min.

Then add the secondary antibody and incubate at 60 rpm for 1 h

After incubation, wash three times with TBSD, each time for 10 min.

Prepare the developer according to the instructions and proceed with development.

After development, add TBSD and store in a 4°C refrigerator.

### Step5. Internal Reference Incubation

Prepare the internal reference primary and secondary antibodies according to the instructions .

Add primary antibody and incubate for 1 hour

After incubation, wash three times with TBSD, each time for 10 min.

Then add the secondary antibody and incubate at 60 rpm for 45 min.

After incubation, wash three times with TBSD, each time for 10 min.

Prepare the developer according to the instructions and proceed with development.

After development, add TBSD and store in a 4°C refrigerator.

## 5. Cell passaging

1. Observe the status of the Hela cells to be passaged under a microscope. Passage can only be performed when the cell confluence reaches more than 80%.

2. Wipe the clean bench with 75% alcohol and irradiate with UV light for 15-30 minutes. Remove 0.25% trypsin, PBS, and DMEM medium containing 10% fetal bovine serum and 1% double-antibody from the refrigerator and return them to room temperature. Remove the sealing film from the PBS and trypsin bottles and sterilize the stoppers and bottle caps over the flame of an alcohol burner.

3. Remove the HeLa cells to be passaged, remove the old culture medium with a pipette, add 3-5 mL of PBS, and gently shake to wash the cells twice.

\*Be careful not to put the nozzle into the tube when adding liquid, and do not blow on the cells.

4. Add an appropriate amount of trypsin to cover the entire cell culture surface ( 1-2 mL for a 25 cm2 cell culture flask ) and shake gently.

\*30 seconds after adding trypsin, aspirate the trypsin and wait for the trypsin remaining in the culture medium to digest the cells; when the cells are about to become round under the microscope, stop digestion and aspirate the trypsin;

\*Alternatively, do not remove the trypsin and observe the cell status under the microscope until the cells are separated and the boundaries are clear.

5. Add 3-5 ml of DMEM medium containing fetal bovine serum/ double antibody and pipette against the wall of the cell culture dish to prepare a cell suspension.

\*When adding culture medium, do not add it to the side where cells are attached. Add it to the corners first and then pipette around to avoid excessive foaming.

\*The blowing position should be even, from top to bottom, from left to right, in sequence to ensure that the cultured cells in each part can be blown, until the cells in the sheet have been dispersed into small clusters of cells or single cells, then stop blowing.

6. Passaging: After all cells have fallen off the culture dish, pipette the cell suspension to 1/3 of the original volume, and fill the remaining 2/3 with new DMEM medium containing fetal bovine serum / double antibody .

7. Place in a CO2 incubator and culture for 18-24 h.

## 6. qPCR steps

The qPCR here is mainly divided into three steps

RNA collection → RT-PCR → qPCR

### Step1. Cell Blow-off

1. Discard the old culture medium and add 500ul BufferRL to blow the cells and collect them without digesting them from the cell plate.

2. First collect the old culture medium into a centrifuge tube. After trypsin digestion for 30 seconds, add culture medium to stop digestion, and collect cells by pipetting. Centrifuge, add 500ul Buffer RL, pipette, and vortex until there are no obvious cell clumps.

### Step2. RNA Extraction

1. Transfer the sample to a gDNA column and centrifuge at 12,000 rpm (13,400 x g) for 30 seconds. Discard the column and collect the filtrate.

2. Add 0.5 times the volume of body fluid anhydrous ethanol to the concentrated solution and mix thoroughly

(After adding the solution , the solution will become turbid or flocculated . This is a normal phenomenon. After shaking and mixing, you can proceed directly to the next step.)

3. Transfer the entire mixture from step 2 to the RNA column and spin at 12,000 rpm. Discard the mixture .

4. Add 700 μl of Buffer RW1 at 12,000 rpm for 30 seconds and discard the mixture.

5. Add 700 μl of Buffer RW2 at 12,000 rpm for 30 seconds and discard the mixture.

6. Add 500 μl of Buffer RW2 at 12,000 rpm for 2 minutes (carefully remove the mixture to avoid contact with the filtrate).

7. Empty the column and spin at 12,000 rpm for 1 minute. Let it sit for 1-2 minutes.

8. Transfer the sample to a high-centrifuge tube ( the kit comes with 3% E). Add 50 μl of enzyme-free water to the center of the column , let it stand for 5 minutes, and then elute at 12,000 rpm for 1 minute. (If the yield is not high, try a second elution.)

9. If not used immediately, store in a refrigerator at -80 °C .

### Step3. RT-PCR

Operate in a fume hood,

gDNA The reagents were stored in a -20° C refrigerator.

1. Preparation system:

* 5x gDNA clean Reaction Mix 2μl
* Total RNA 1μg
* RNase free water 10μl
* 42°C 2 min
* Remove from heat at 4°C

2. Reverse transcription reaction

1) Preparation system (20ul)

* reaction solution 10 μL
* 5x EvM-MLV RT Reaction Mix(Yellow) 4ul
* RNase-free water 6 μL

2) PCR Procedure:

Mix the RCR system at 4°C

### Step4. qRCR

When plating cells, perform three replicate wells.

Preparation of qPCR system: (You can add enzyme-free water and primers first, then add cDNA, and then add 2\*mix)

* Primer (10 μm) 0.7 μL
* cDNA 1.75 μL
* 2xMix 17.52μL
* Water 14.33μL

The internal reference microglobulin and GADPH are only used as blank groups.

Each sample was divided into 3 portions of 20 and added to 96 wells

When applying the anti-steaming film, do not touch the center of the film. After tearing off the film, stick the sticky side to the 96-well plate, then buckle the white paper, scrape it flat with the plate, and then tear off the excess parts on both sides.

Before loading, centrifuge at 800 rpm for 2 minutes.Peel off the white paper, leave the film , and feed into the machine.

### Step5. qPCR data processing

Quantitative PCR was performed on a real-time PCR system , and data were collected and analyzed. All samples were prepared and replicated three times. Experiments were performed at least three times independently. To calculate relative concentrations, ΔCT values were obtained for all samples. ΔCT is a normalized, relative gene expression level. This is achieved by normalizing for the degradation effect of siRNA. Normalized expression for each sample was obtained by subtracting the CT value of the siRNA from the same sample and designated as ΔCT. This value was then converted to a comparative fold change by performing the log(2 − ( ΔCT )).

## 7. siRNA transfection experiments

### Step1. Cell seeding:

Adherent cells: Taking 293T cells as an example, 0.4x10\* cells were seeded into a six-well plate containing 2 mL of complete medium to achieve a cell density of 50-80% during transfection.

Suspension cells: Taking THP-1 cells as an example, seed 0.5x10 cells into the wells of a six-well plate containing 2 mL of complete culture medium.

**Note:**

1: The initial inoculation number of different cells depends on the different cell size, cell type, cell growth rate, culture time and experimental purpose. The number of cells inoculated in each well should be as similar as possible and the cells should be evenly distributed.

2: Suspension cells, immune cells, and primary cells are generally difficult to transfect due to their inherent characteristics. You can try to improve the transfection effect by adjusting the amount of siRNA and transfection reagent, using different transfection reagents, and standardizing the experimental operation as much as possible. If there is no obvious improvement, you can switch to electroporation or other methods.

### Step2. siRNA rapid staining

Preparation of siRNA premix: Take a sterile enzyme-free EP tube, add 46ul of GA-RNA Buffer and 4ul of 20umol/L siRNA storage solution, and mix well by pipetting.

To prepare the sIRNA -GA rapid dye complex , add 7.5 μL of GA-RNA Reagent to the EP tube containing the sIRNA premix and pipette thoroughly to mix for more than 20 times, or vortex for 2-35 seconds.

**Note:**

1. The transfection complex is stable at room temperature for 8 hours. Do not freeze the siRNA -GA transfection complex.

2. The RNA/GA-RNA Reagent ratio is a key factor in the encapsulation of the GA-RNA Transfection Reagent. If you need to increase or decrease the amount of SIRNA per well, you can increase or decrease the volume of siRNA -GA transfection complex added to the cells accordingly.

Add the siRNA -GA rapid transfection complex to the cell culture medium of the well plate to be transfected and mix gently.

Other necessary special treatments (optional, such as drug addition, etc., determined according to specific experimental conditions),

Place the culture plate in a 37°C CO incubator and culture for 24-96 hours (the culture time is related to the experimental purpose. qPCR is generally performed 24-48 hours after transfection to extract RNA for RT-qPCR testing; Western blotting is generally performed 48-96 hours after transfection to collect cells for Western blotting testing).

Table 4 Recommended dosage for rapid staining of different culture dishes with a final concentration of 40 nmol/L siRNA

| Reagent | 96 well plate | 24 well plate | 12 well plate | 6 well plate |
| --- | --- | --- | --- | --- |
| GA-RNA Buffer(μl) | 2.3 | 11.5 | 46 | 23 |
| 20 μmol/L siRNA storage solution(μl) | 0.2 | 1 | 2 | 4 |
| GA-RNA Reagent(μl) | 0.38 or 0.75 | 1.9 or 3.8 | 3.8 or 7.5 | 7.5 or 15 |

Note: Two different concentrations of GA-RNA Reagent are available for rapid staining . The specific concentration can be selected based on the specific cell type. For most cells, the ideal rapid staining effect can usually be achieved with the lower dose of GA-RNA Reagent .

### Step3. siRNA efficacy testing

RNA level detection: RNA levels should be detected 24-48 hours after siRNA transfection. Detecting changes in target gene RNA levels by qPCR is a widely accepted standard for RNAi experimental results. The optimal detection time varies depending on the cell type. Testing too late may result in poor interference detection efficiency or even failure to detect interference effects.

Protein Level Detection: For protein-coding genes, it is recommended to perform protein detection 48-96 hours after transfection. Because protein expression is affected by many factors, if protein levels are unclear, please test mRNA levels to confirm the effectiveness of the siRNA.

## 8. Preparation and Characterization of Hybrid Delivery Vectors

### Step1. Vaccination

The Lpp-OmpA-eGFP and Lpp-OmpA-Anti GPC3 chimeric systems were selected , with Escherichia coli BL21 as the host bacteria. Escherichia coli that stably expressed eGFP and GPC3 antibodies on the outer membrane (abbreviated as LOE and GPC3 ) were cultured ; at the same time, Escherichia coli expressing eGFP intracellularly was cultured as a positive control, and BL21 was used as a negative control.

**Experimental operation:**

1. Resuscitation of bacterial strains: Remove the glycerol tube from -80℃ and place it on ice immediately.

2. Streak inoculation: Dip the bacterial solution and streak it on the LB plate . Add 100 μg /mL kanamycin to the resistance plate .

3. Culture: Invert the plate and culture in a 37°C incubator for 12-16 hours.

### Step2. Bacterial culture

1. Pick a single, isolated colony with regular morphology and neat edges that has formed on an LB plate after 16 hours of incubation at 37°C . Inoculate the selected bacteria into 10 mL of sterile LB medium supplemented with kanamycin at a final concentration of 50 μg /mL for the LOE , GPC3 , and eGFP strains . Incubate the inoculated bacterial suspension at 37°C in a shaker at 220 rpm for 12-16 hours to allow the bacteria to fully recover and enter early logarithmic growth.

2. Transfer the bacterial suspension from the 12-16 hour pre-culture to fresh LB liquid medium ( supplemented with Kanamycin at a final concentration of 50 μg / mL for the LOE , GPC3 , and eGFP strains ) at a 1:100 (v/v) inoculum ratio . Incubate at 37°C and 220 rpm with constant shaking for approximately 4 hours, until the OD₆₀₀ value reaches 0.6 . Immediately, add IPTG (isopropyl -β-D- thiogalactopyranoside ) to the culture medium to a final concentration of 2 μmol /mL . Adjust the culture conditions to 25 °C and 180 rpm , and continue induction for 16 hours to promote expression and correct folding of the target protein.

### Step3. Membrane Vesicle Preparation

1. Bacteria Collection and Cleaning

The bacterial suspension was divided into 50 mL centrifuge tubes and centrifuged at 8,000× g at 4°C for 3 min (balanced with deionized water before centrifugation). The supernatant culture medium was completely discarded and the centrifuge tubes were placed on ice to maintain a low temperature environment. Then, pre-chilled PBS buffer ( pH 7.4 ) was used. Wash the bacterial pellet twice: add 10 mL of PBS each time to fully resuspend the bacteria, then centrifuge again at 8,000× g and 4°C for 3 min , discarding the supernatant to ensure removal of residual culture medium components and secreted metabolites.

2. Ultrasonic cell disruption

Resuspend the washed cells in an appropriate amount of PBS and disrupt them using an ultrasonic disruptor . Keep the centrifuge tube in an ice-water mixture for temperature control throughout the process. Set the ultrasonic program to 5 seconds on, 5 seconds off , for a total of 10 minutes . Ensure that the probe is submerged below the liquid level and does not contact the tube wall to achieve efficient cell lysis while avoiding sample overheating and foaming.

3. Removal of Cell Debris

The lysate after disruption was centrifuged at 17,000× g and 4°C for 30 min (precisely balanced before centrifugation) to separate and remove unbroken cells and large membrane fragments. The supernatant was carefully collected and placed on ice. This supernatant is the crude extract containing vesicles.

4. Ultra-high-speed centrifugation to enrich vesicles

The supernatant was filtered through a 0.22 μm sterile filter to remove residual impurities. Aliquots were then dispensed into ultracentrifuge tubes, accurately balanced using a 1/10,000 analytical balance (each tube was accurately balanced to within ±0.01 g ). Ultracentrifugation was performed at 170,000× g and 4°C for 30 min . A translucent vesicle pellet was visible at the bottom of the tube .

5. Vesicle Resuspension and Storage

Prepare PBS buffer filtered through a 0.22 μm filter . After centrifugation, quickly discard the supernatant to avoid disturbing the pellet. Gently resuspend each pellet in 1 mL of pre-chilled PBS by pipetting. Combine the resuspensions in pre-chilled 1.5 mL centrifuge tubes. Keep on ice throughout the entire process. Finally, aliquot and store at 4 ° C .

### Step4. Nanoflow cytometry

Detecting properties such as membrane vesicle size, particle number, and protein fluorescence intensity

1. System startup

1) Pre-startup inspection: Check the level of the wash solution (>10mL) and the height difference between the sheath solution and the waste solution (20-30cm).

2) Turn on the computer and run the NF Profession2.0 software. When you hear two "beep-- beep-- " sounds, it means the device is connected successfully.

3) System initialization: Click Start Up. The camera, laser and air pump are now turned on.

4) Flow Initialization: Start the flow system by clicking Sheath Flow > StartUp . Place 150 L of ultrapure water on the sample holder . Click Sample > Boosting and allow the flow to initialize for 5 minutes. After initialization, click Sample > Unload.

5) Eliminate bubbles: Observe the tubing inside the right door of the instrument to see if there are any bubbles remaining. If so, click Sheath Flow > Empty Sheath. Once the bubbles are eliminated, click Sheath Flow > Purge. Once the bubbles are eliminated, the Sheath Flow status will automatically switch to Normal .

Routine quality control of nanoflow cytometry

2. Concentration standard detection

1) Sample loading : Load concentration standard (250nm SiNPs, 100uL), Sample--Boosting, time 1min.

2) Parameter Settings: In Samp.Inf , select "250nm Std FL SiNPs " and the default parameters are Laser power: 20mW, SS Decay: 0.2%. Also, enter a dilution factor of 100. The sample name can be manually modified.

3) Sampling: Sample--Sampling, click A in the toolbar and select Large signal to automatically set the threshold; click Auto Sampling and enter 1.0 in the box to the right of Sampling SET to fix the Sampling pressure at 1.0 kPa; wait for the Sampling pressure to stabilize at 1.0 kPa, then click Time to Record to collect data: After data collection is completed, the collection mode automatically jumps to Buffer. Click the Save button in the pop-up box to save the data as Nfa File; Unload the sample.

4) Capillary cleaning: Load the capillary with cleaning solution (150 μL), sample for 1 minute, and then unload. Remove any residual cleaning solution from the capillary tip with ultrapure water (150 μL).

3. Particle size standard testing

1) Kamiyoshi : Load particle size standard product (Silica Nanospheres68~155nm, 100uL), Sample--Boosting. Time 1min.

2) Parameter setting: Select "68-155S16M-Exo" in Samp.Inf . The sample name can be modified manually.

3) Sampling: Sample--Sampling, click the toolbar and select Small signal, automatically set the threshold: Fix the Sampling pressure to 1.0kPa: Click Time to Record to collect data: Click the Save button in the pop-up box to save the data as Nfa File; Unload the sample.

4) Capillary cleaning: Load the capillary with washing solution (150 μL), sample-boosting for 1 minute, and unload. Use ultrapure water (150 μL) to remove any residual washing solution from the capillary tip.

4. Blank control test

1) Loading : Load blank control (sample dilution solution, such as PBS), Sample--Boosting, time 1 min.

2) Parameter setting: Select "68-155S16M-Exo" in Samp.Inf , and pay attention to modifying the sample name (the detection conditions of the blank control should be consistent with those of the sample).

3) Sampling: Sample--Sampling, click on the toolbar

And select Small signal, automatically set the threshold: Fix the Sampling pressure to 1.0kPa: Click Time to Record to collect data: After data collection is completed, click the Save button to save the data as Nfa File: Unload the blank control.

5. Sample testing

1) Loading : Load sample (100uL), Sample--Boosting, time 1min.

2) Parameter setting: Select "68-155S16M-Exo" in Samp.Inf , change the sample name and enter the dilution factor.

number.

3) Sampling: Sample--Sampling, click the toolbar and select Small sigal to automatically set the threshold; fix the Sampling pressure to 1.0kPa; wait for the Sampling pressure to stabilize at 1.0kPa, click Time to Record to collect data; after data collection is completed, click the Save button to save the data as Nfa File: Unload the sample.

4) Capillary cleaning: Load the capillary with washing solution (150 μL), sample-boosting for 1 minute, and unload. Use ultrapure water (150 μL) to remove any residual washing solution from the capillary tip.

### Step5. Preparation of EV- LNP Hybrids

LNP components of formula 1273 , molar ratio: DOTAP: cholesterol :DSPC:PEG- lipid = 50:38.5:10:1.5

Liposome concentration ratio ( Formula 1273 )

| Components | content | Mv | Weigh the mass | concentration | Weigh the volume |
| --- | --- | --- | --- | --- | --- |
| HUO | 50% | 709 | 0.008863 g | 0.1 g/mL | 88.63μL |
| D0PE | 10% | 744.034 | 0.00186 g | 0.1 g/mL | 18.60μL |
| cholesterol | 37.5% | 386.65 | 0.003635 g | 0.02 g/mL | 181.75μL |
| DSPEPEG2000 | 2.5% | 2000 | 0.001250 g | 0.01 g/mL | 125μL |

**Steps:**

1. HUO (SM-102) is a viscous liquid. First, add 1 ml of anhydrous ethanol to the HUO (SM-102) centrifuge tube.

2. Liposome Preparation (Concentration: 12.5 mM ): Use a micropipette to accurately weigh the appropriate volumes of cationic carrier, amphiphilic phospholipid bilayer , cholesterol, and DSPE-PEG2000 into a 2 mL centrifuge tube . Add DMSO -dissolved coumarin 6 (C6) fluorescent dye for labeling to a final concentration of 160 μmol/L . Make up the volume to 2 mL with 100% ethanol .

3. Mix the citric acid buffer and LNPs at a ratio of 3:1 by pipetting and incubate at room temperature for 10 min .

4. Vortex vigorously for 1 minute , then place in a 10 Kd dialysis bag and dialyze against PBS buffer at 4 °C in the dark to remove unlabeled free C6 . Store in a 4 °C refrigerator until ready for use.

For fluorescent labeling of EVs , DID (excitation / emission: 644/665 nm ) was selected as the fluorescent dye. To 200 µL of purified EVs (concentration approximately 4 × 10 10 particles /mL ), DID solution was added to the solution to a final dye concentration of 40 µmol/L . The solution was incubated with rotation at room temperature in the dark for 1 hour. The solution was then dialyzed against PBS buffer at 4°C in a 10Kd dialysis bag in the dark to remove unlabeled free DID dye. The solution was then refrigerated at 4°C until ready for use.

The prepared EVs and LNP liposome solutions were diluted to 4×10 9 particles /mL and thoroughly mixed at a particle concentration ratio of 1 : 1 . They were immediately frozen in liquid nitrogen for 1 min , then completely thawed in a 37 °C water bath and incubated for about 5 min . After repeating the operation three times , the hybrid particles were homogenized using an ultrasonic disruptor . The ultrasonic program was set to 5 s on , 5 s off , for a total of 5 min . The mixture was then placed at 4 °C for use.

Nanoflow cytometry was used to detect the properties of EV-DID , LNP-C6 and hybrids.

### Step6. Characterization

1. Characterization with Zeiss Confocal Microscope

20 μl of sample was dropped onto a glass slide, which was then covered with a coverslip and photographed using a Zeiss confocal microscope under a 60× oil objective lens .

2. Western Blot Detection of Vesicle Proteins :

Protein electrophoresis was performed on 1.5 mm thick SDS-PAGE gels (12% separating gel concentration, 5% stacking gel concentration). An appropriate amount of purified vesicle sample was mixed with 5 × SDS - PAGE protein loading buffer at a 4:1 volume ratio . After thorough vortexing, the mixture was heated in a boiling water bath for 5 minutes to fully denature the proteins.

After the sample cools, briefly centrifuge to collect the condensate on the tube wall. Add sufficient 1× electrophoresis buffer to the electrophoresis tank, loading 20 μL of sample per well. Add 5 μL of prestained protein molecular weight standards to adjacent lanes as a reference. Run two gels simultaneously. Electrophoresis parameters are as follows: an initial voltage of 80 V to allow the sample to migrate and compact into a narrow band in the stacking gel. When the bromophenol blue indicator reaches the separating gel interface, increase the voltage to 150 V and continue running until the bromophenol blue indicator migrates to the bottom edge of the gel, at which point the electrophoresis is terminated.

Take a 4.5 x 8.5 cm PVDF membrane and soak it in anhydrous methanol for 1 minute to activate the hydrophobic groups. Then, transfer it to transfer buffer for 5 minutes. Assemble the SDS-PAGE gel and pretreated PVDF membrane in a " sandwich " configuration in a transfer cassette, strictly adhering to the principle of facing the negative electrode with the gel side facing the positive electrode and the membrane side facing the positive electrode. Remove any air bubbles and place the membrane in the transfer chamber .

After transfer , transfer the membrane to a high-efficiency blocking buffer and block on a shaker at room temperature for 10 minutes. After blocking, prepare the primary antibody working solution : Dissolve 1 g of skim milk powder in 20 mL of TBST buffer. Add 2 μL of mouse His monoclonal antibody and mix thoroughly. Immerse the membrane in the primary antibody working solution and incubate at room temperature for 30 minutes before transferring to a 4°C refrigerator for overnight incubation.

Soak the membrane in methanol for 1 minute to activate it, then soak it in transfer buffer for 5 minutes . Transfer the membrane using a transfer apparatus , ensuring the gel is close to the negative electrode. After transfer , block the membrane with a high-efficiency blocking solution for 10 minutes . Prepare the primary antibody: 20 ml TBST + 1 g skim milk powder + 2 μL His antibody. After blocking, incubate with the primary antibody for 30 minutes and refrigerate at 4 °C overnight.

Another SDS-PAGE gel was stained with Coomassie Brilliant Blue and destained, and the whole protein band was photographed using a gel imaging system as a control.

After primary antibody incubation, remove the membrane and wash it six times with TBST buffer for 10 minutes each to completely remove any unbound primary antibody. Prepare the secondary antibody working solution : Dissolve 1 g of skim milk powder in 20 mL of TBST buffer, then add 2 μL of horseradish peroxidase- conjugated goat anti-mouse IgG secondary antibody. Incubate on a shaker at room temperature for 1 hour, then wash the membrane six times with TBST for 10 minutes each . Apply the luminescent solution evenly to the membrane surface , allow to react for 1 minute, and remove any remaining liquid by aspiration. Acquire the signal using a chemiluminescence imaging system.

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{% block title %}Results{% endblock %}
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![iGEM lead-img](https://static.igem.wiki/teams/5844/lead/dut-china-7973.webp)

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[Abstract](#abstract)
[Drug Platform](#drug-platform)
[Delivery Carrier](#delivery-carrier)
[Target Application](#target-application)

## Abstract

This project successfully constructed a modular mRNA drug platform for hepatocellular carcinoma: In terms of platform construction, we generated efficient artificial 5'UTR sequences through multiple rounds of model iterative optimization, and screened out 30× exCAG-based sequences Molecular switching of repeats to achieve tumor-specific expression regulation; In terms of delivery system, we successfully exhibited GPC3 antibodies on the surface of artificially prepared bacterial outer vesicles and performed heterozygous characterization of EVs and LNPs by fluorescence staining technology, achieving a heterozygous efficiency of about 80%; Finally, we verified the "dual regulation" strategy at the core of the platform at the functional level, that is, the siRNA was used to successfully degrade the proto-oncoprotein TGF-β (efficiency >70%), and confirmed the feasibility of "up-regulating detoxification protein and down-regulating proto-oncoprotein at the same time" through a dual fluorescence reporting system. The platform integrates efficient expression, precise targeting, and synergistic regulation, laying a solid foundation for the development of next-generation mRNA therapeutics.

## 1. Drug platform design module

### 1.1 5’UTR Optimization Module

The 5′ untranslated region (5′UTR) plays a key role in ribosomal recruitment, regulation of translation initiation, and maintenance of mRNA stability. In order to obtain the manually optimized design of the 5’UTR sequences, we selected two models, UTRGAN and UTR-Insight, and iteratively optimized them based on the model characteristics and operating requirements. A total of 39 sequences were output in three batches, and 35 were verified.

In terms of characterization, we selected luciferase as the reporter protein and designed luciferase-expressing RNA (Luc-UTR RNA) to evaluate the regulatory efficiency of different 5′UTR variants.

In order to further characterize our 5’UTR effect, we collected the NCA-7d-5'UTR sequence, which is a highly expressed endogenous 5’UTR, and the Albumin-5'UTR sequence, which is an albumin gene-derived 5’UTR, both are naturally occurring 5’UTR sequences in nature and have been used in mRNA therapy research. We want to compare it with the regulation effect of our self-designed 5’UTR to better evaluate our optimization results.

![Characterization of Regulatory Efficiency for the First Set of 5'UTR Sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result1.webp)

**Figure 1. Characterization of Regulatory Efficiency for the First Set of 5'UTR Sequences** Sequence numbers 1–10 represent nine differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, while NC serves as a blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).

![CCIC event photo 1](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result2a.webp)
![CCIC event photo 2](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result2b.webp)

**Figure 2. Efficiency Characterization of the Second Set of 5'UTR Sequences** Sequence numbers 11-24 represent 14 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, while NC serves as a blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).

![The third batch of 5'UTR sequence regulatory efficiency characterization](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result3a.webp)

**Figure 3. The third batch of 5'UTR sequence regulatory efficiency characterization.** Numbers 26-39 represent 14 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized sequence, the NCA group uses the NCA-7d-5'UTR sequence, the Alb group uses the Albumin-5'UTR sequence, and NC serves as the blank control without transfection of RNA. Luciferase activity was measured and is presented as Relative Light Units (RLU).

To further compare the regulatory effects of three batches of 5'UTR to evaluate our model optimization results, we normalized the fluorescence data for each batch according to the PC group.

![Comparative Characterization of Regulatory Efficiency Among Three Batches of 5'UTR Sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result4a.webp)

**Figure 4. Comparative Characterization of Regulatory Efficiency Among Three Batches of 5'UTR Sequences.** Numbers 1–39 denote 34 differently designed 5'UTR sequences. The PC group uses Pfizer's publicly available artificially optimized 5'UTR sequence, the NCA group uses the NCA-7d-5'UTR sequence, the Alb group uses the Albumin-5'UTR sequence, and NC represents the blank control without transfection of RNA. Fluorescence intensity data were measured at 560 nm wavelength using a microplate reader. The vertical axis represents the ratio of fluorescence intensity in the experimental group to that in the PC group.

![Comparison of Output Quality Among Three Batches of 5'UTR Models](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result5a.webp)

**Figure 5. Comparison of Output Quality Among Three Batches of 5'UTR Models** Fluorescence intensity data were measured at 560 nm wavelength using a microplate reader. The vertical axis represents the ratio of fluorescence intensity in the experimental group to that in the PC group.

According to the analysis of experimental data, the design quality of the third batch of 5'UTR is improved compared with the previous two batches, and the regulatory effect of the No. 37 5'UTR sequence is better than that of the two high in nature Expressing 5'UTR, it has application potential. So far, the iteration of our UTR optimization model has achieved certain results.

### 1.2 Molecular switch design module

While mRNA therapeutics have shown promise in vitro and in vitro experiments, they still face several challenges, particularly off-target effects and insufficient specificity. In order to avoid the cytotoxicity that protein regulatory modules may cause in normal cells and to solve the above key problems, we design and optimize two types of RNA molecular switches: exCAG sequence-based switches and Toehold switches.

The Toehold switch is an artificially designed RNA riboswitch that hides the translation initiation region in the mRNA secondary structure and initiates translation only when a specific trigger RNA is present. At present, Toehold switches have been extensively studied in prokaryotic systems, but there are still deficiencies in eukaryotic systems. In this study, a series of eukaryotic Toehold switches were constructed and validated based on existing data, with luciferase as the reporter gene. The regulatory ability of different switches was evaluated by comparing the fluorescence intensity with and without miR-21.

![Validation of the self-designed toehold sequence for regulating protein expression](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result6.webp)

**Figure 6. Validation of the self-designed toehold sequence for regulating protein expression.** Luciferase activity was measured and presented as Relative Light Units (RLU). Groups C1, E1, I1, K1, and T1 were transfected with mRNA containing only the toehold switch sequence and luciferase sequence. Groups C2, E2, I2, K2, and T2 were transfected with mRNA and miR-21 mimics. Groups C3, E3, I3, K3, and T3 were transfected with mRNA and miR-21 inhibitors. The PC group was transfected with mRNA expressing luciferase without the switch sequence, while the NC group received no transfection. Statistical significance was assessed using ANOVA with Bonferroni-corrected multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

The experimental results show that the toehold switch from the literature source has a lower opening level, while the toehold switch generated by the model has a higher leakage expression and poor regulatory effect. Therefore, we decided to continue to optimize the Toehold switch generation model from the dry experimental dimension, and explore other feasible molecular switch designs.

The exCAG switch consists of a miR-21 binding region and a repressive sequence, located downstream of the mRNA polyA tail. The repression region consists of repeated CAG sequences that silence the ORF region by inducing RNA gelation. When the concentration of miR-21 in cells is high, its complementary region pairs with miR-21 and recruits the RISC complex to cleave the inhibitory sequence, thereby degelating RNA, allowing ORF to be expressed normally and achieving cell selectivity.

We designed three mRNAs with mcherry sequences as reporters (CAG30 mRNA, CAG10 mRNA, CAG3 mRNA), in The posterior insertion of the polyA tail of the three RNAs adds the miR-21 binding sequence, and the CAG sequence with different repeat numbers is inserted as the repression sequence (30×, 10×, 3×). to explore the optimal exCAG repetitions and improve switch selectivity.

![Fluorescence detection results of protein expression regulation by 30xCAG, 10xCAG, and 3xCAG sequences](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result7.webp)

**Figure 7. Fluorescence detection results of protein expression regulation by 30xCAG, 10xCAG, and 3xCAG sequences.** mCherry fluorescence was detected by flow cytometry and presented as Mean Fluorescence Intensity (MFI). The NC group was not transfected with any RNA. Statistical significance was assessed using ANOVA combined with Bonferroni's multiple comparison test (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

The experimental results showed that the control group transfected with CAG10 mRNA and the experimental group transfected with CAG10 mRNA and miR-21 mimic were detected by flow cytometry using B610 channels. There was no significant difference in mCherry fluorescence intensity (p>0.05). However, the control group transfected with CAG3 mRNA and the experimental group transfected with CAG3 mRNA and miR-21 mimic showed a significant difference in fluorescence intensity (p<0.01). Similarly, the control group transfected with CAG30 mRNA and the experimental group transfected with CAG30 mRNA and miR-21 mimic also showed a significant difference in fluorescence intensity (p<0.001). Based on the experimental results, we guessed that the better CAG repeat number might be around or above 30. As for the regulatory effect of CAG3 mRNA, it may be caused by other mechanisms, which needs to be further explored.

In order to further evaluate the regulatory effect of the two types of switches on protein expression under different designs, and to screen out the optimal scheme, we compared the induction folds of the above switch designs.

![Induction ratio for different switch designs](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result8a.webp)

**Figure 8. Induction ratio for different switch designs** Induction ratio = (Fluorescence intensity measured with mRNA + triggerRNA / Fluorescence intensity measured with mRNA alone)

Comparing the induction folds and switch levels of the above five switches, we will select a molecular switch based on the 30xexCAG sequence as part of our molecular platform design, thereby improving the safety of our drugs.

## 2. Delivery carrier design

Lipid nanoparticles (LNPs) are the core delivery system of current nucleic acid drugs, which can effectively overcome the multiple barriers of mRNA in vitro and vitro, and have the advantages of low immunogenicity, high encapsulation efficiency, organ targeting, and efficient intracellular delivery. GPC3 is a glycoprotein associated with liver cancer that is highly expressed in 70–80% of patients with hepatocellular carcinoma, especially in poorly differentiated tumors, and almost non-existent in normal adult liver tissue.

Based on this, we intend to confer the specific recognition ability of HCC cells by conjugating GPC3 antibody on the surface of LNP. The specific strategy consists of three steps: (i) presentation of GPC3 antibodies using the Lpp-OmpA Presentation System in E.coli; (ii) preparation of extracellular vesicles (EVs) that surface present GPC3 antibodies; (iii) It was hybridized with LNPs to prepare LNP-EV hybridized vectors carrying GPC3 antibodies on the surface.

In terms of protein presentation, we successfully demonstrated eGFP and GPC3 single-stranded antibodies on the surface of BL21 E.coli, respectively, and characterized them by nanoflow cytometry and Western blot experiments.

![Nanostream flow cytometry characterization of fluorescence intensity in eGFP-presenting EVs](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result9.webp)

**Figure9. Nanostream flow cytometry characterization of fluorescence intensity in eGFP-presenting EVs** a: Test sample: eGFP-presenting EVs b: Test sample: EVs derived from wild-type BL21.

![Western blot results for EVs displaying eGFP and GPC3 monoclonal antibodies on their surfaces](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result10a.webp)

**Figure 10. Western blot results for EVs displaying eGFP and GPC3 monoclonal antibodies on their surfaces.** Lane 1 shows EVs produced by wild-type BL21 as a negative control. Lanes 2 and 3 display EVs presenting eGFP protein on their surfaces. Lane 4 shows EVs presenting GPC3 monoclonal antibodies on their surfaces. The expected molecular weights for both the Lpp-OmpA-eGFP fusion protein and the Lpp-OmpA-GPC3 monoclonal antibody fusion protein are approximately 42 kDa. The protein loading amount was 5 ng.

In terms of LNP-EVs hybridization, we performed DID staining of BL21-derived EVs and coumarin C6 staining of LNPs, realized the heterozygosity of the two by freeze-thaw method, and detected the hybridization efficiency by nanoflow cytometry.

![Fluorescence Characterization of EVs-LNP Hybrids](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result11.webp)

**Figure11 Fluorescence Characterization of EVs-LNP Hybrids** a: BL21-derived EVs stained with DID; b: LNP stained with coumarin C6; c: EVs-LNP hybrid staining; d: Fluorescence intensity of unstained BL21-derived EVs

The heterozygous effect was well characterized, and the fluorescence detection rate of hybrid vesicles could reach about 80%. The fluorescent labeling rate increased after hybridization, which may be caused by the migration of dye molecules during hybridization freeze-thawing.

## 3. Target application design

Based on the existing m-PROTAC strategy, we designed our protein regulatory element, which consists of three modules, namely the Trim21 protein, the ligation peptide, and a short peptide that can specifically bind to the target protein. The common fusion protein of these three modules can specifically bind to the target protein and reduce the content of related oncogenic proteins in hepatocellular carcinoma cells by mediating the ubiquitination degradation pathway, thereby inhibiting cancer.

At the same time, hepatocellular carcinoma (HCC) is a highly heterogeneous and complex malignant tumor, and its occurrence and development involves abnormal activation of multiple signaling pathways, making it difficult for any single-target drug to achieve durable and effective tumor control.

Based on this, we added protein expression elements to the protein regulation module. It is hoped that the expression of tumor suppressor gene-related proteins can be improved while degrading the proteins expressed by the original oncogene in hepatocellular carcinoma cells, thereby improving the anti-cancer effect of drugs. Here we select TGF-β protein as our protein degradation target and HNF4α protein as our protein expression target.

To verify the feasibility and effectiveness of our protein regulatory elements, we conducted the following experiments:

First, we designed siRNAs targeting bases 48, 339 and 642 of TGF-β protein mRNA to knock down TGF-β protein gene expression at the RNA level. The changes of TGF-β protein content and mRNA content in hepatocellular carcinoma cells after transfection with siRNA were detected by RT-qPCR and Western blot assays to verify the feasibility of our protein degradation target selection.

![Analysis of RT-qPCR results from TGF-β gene siRNA silencing experiments](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result12.webp)

**Figure 12. Analysis of RT-qPCR results from TGF-β gene siRNA silencing experiments.** Data from three groups (siRNA1, siRNA2, siRNA3) were obtained from samples transfected with siRNAs targeting positions 642, 48, and 339 of the TGF-β mRNA, respectively. Quantitative PCR was performed on a real-time PCR system for data collection and analysis. All samples were prepared and replicated three times. The experiment was performed independently at least three times. To calculate relative concentrations, CT values were obtained for all samples. ΔCt represents a normalized, relative gene expression level. This normalization was achieved by standardizing the degradation effect of TGF-β siRNA. Normalized expression for each sample was obtained by subtracting the CT value of the internal reference gene (GAPDH) from the same sample, designated as ΔCt. Compare the ΔCt of the experimental group with the ΔCt of the control group. Formula: ΔΔCt = ΔCt(siRNA) individual - ΔCt(siRNA) mean. Then convert this value to a comparative fold change by performing log(2 −(ΔΔCT)). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

![Western Blot results for TGFβ gene silencing by siRNA](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result13.webp)

**Figure 13. Western Blot results for TGFβ gene silencing by siRNA.** Lanes siRNA1, siRNA2, and siRNA3 correspond to samples transfected with siRNA targeting positions 642, 48, and 339 of TGFβ mRNA, respectively. Lanes Untreated1–Untreated3 on the right serve as blank controls. Cells were lysed and analyzed 48 hours after siRNA transfection. Protein loading was 20 μg.

![Western Blot Gray Value Analysis of TGFβ Gene Silencing by siRNA](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result14.webp)

**Figure 14. Western Blot Gray Value Analysis of TGFβ Gene Silencing by siRNA** Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

Based on the RT-qPCR experimental analysis, the degradation rate of siRNA of the three TGF-β reached 70% or more, and Western blot was also used Experimental grayscale analysis also showed that we successfully silenced the TGF-β gene. It was proved that the protein expression of this target could be artificially regulated.

To validate the necessity of enhancing HNF4α protein expression, we designed a Western blot assay to compare HNF4α protein levels between hepatocellular carcinoma cells and other cell types. Results demonstrated reduced HNF4α expression in hepatocellular carcinoma tissues, confirming the feasibility of selecting this protein expression target for our study.

![Western Blot results for intracellular HNF4α protein expression](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result15.webp)

**Figure15. Western Blot results for intracellular HNF4α protein expression.** The left three lanes represent proteins extracted from 293T cells, while the right three lanes represent proteins extracted from HepG2 cells. Cells were lysed and analyzed 48 hours after siRNA transfection. Each sample contained 20μg of protein.

![Analysis of grayscale values in Western Blot for intracellular HNF4α protein content detection](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result16.webp)

**Figure16. Analysis of grayscale values in Western Blot for intracellular HNF4α protein content detection** Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

Results demonstrated reduced HNF4α expression in hepatocellular carcinoma tissues, confirming the feasibility of selecting this protein expression target for our study.

To verify the feasibility of upregulation and downregulation of two different target proteins at the same time, we designed siRNAs targeting eGFP fluorescent proteins (eGFP siRNAs) and those that can stably express eGFP/Luc, respectively iferase mRNA (eGFP mRNA and Luc mRNA). The feasibility of this strategy was verified by transfecting eGFP mRNA into cells first, and then co-delivering eGFP siRNA and Luc mRNA into cells.

![Fluorescence detection results of eGFP siRNA and luc mRNA co-delivery experiment](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result17a.webp)

**Figure 17: Fluorescence detection results of eGFP siRNA and luc mRNA co-delivery experiment** a: EGFP fluorescence intensity detected by flow cytometry using the B525 channel, presented as Mean Fluorescence Intensity (MFI) b: Fluorescence intensity measured at 560 nm wavelength using a microplate reader, presented as Relative Light Units (RLU). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

The experimental results showed that the green fluorescence intensity of the experimental group transfected with eGFP siRNA was significantly different from that of the control group without siRNA transfection (p<0.0001). Under the same experimental conditions, there was no significant difference in fluorescence intensity at 560 nm wavelength between the two groups (p>0.05). This indicates that we successfully expressed Luc while reducing eGFP expression using siRNA, demonstrating the feasibility of simultaneously upregulating and downregulating two different target proteins.

To evaluate the application potential of the drug, we designed siRNAs (eGFP siRNA and Luc mRNA) that target eGFP fluorescent proteins/Luc iferase, respectively , and those capable of stable expression of eGFP, respectively /Luciferase mRNA (eGFP mRNA and Luc mRNA). The feasibility of this strategy was verified by transfecting eGFP mRNA and Luc mRNA into cells first, and then delivering eGFP siRNA and Luc siRNA into cells.

![Fluorescence detection results of eGFP siRNA and luc siRNA co-delivery degradation experiment](https://static.igem.wiki/teams/5844/pages/wetlab/dut-china-result18a.webp)

**Figure18. Fluorescence detection results of eGFP siRNA and luc siRNA co-delivery degradation experiment** a: EGFP fluorescence intensity detected by flow cytometry using the B525 channel, presented as Mean Fluorescence Intensity (MFI) b: Fluorescence intensity measured at 560 nm wavelength using a microplate reader, presented as Relative Light Units (RLU). Statistical significance was assessed using ANOVA with Bonferroni correction for multiple comparisons (\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001; mean ± standard deviation).

The experimental results showed that the green fluorescence intensity of the experimental group transfected with eGFP siRNA and Luc siRNA was significantly different from that of the control group without transfected siRNA (p<0.0001) , and under the same experimental treatment, there was a significant difference in fluorescence intensity between the two groups at 560nm wavelength (p<0.001), indicating that we used eGFP siRNA and Luc siRNA successfully reduced the expression of eGFP and Luciferase at the same time, proving the feasibility of our strategy of down-regulating the expression of multiple proteins at the same time, and laying the foundation for further experimental exploration.

In future work, we plan to integrate the degradation and expression modules into a single RNA construct, transfect them into HCC cells, and evaluate protein level changes to further confirm therapeutic effects. We expect this drug to complement as a molecularly targeted drug in combination therapy and to have a synergistic effect with immunotherapy drugs. At the same time, we emphasize modular construction in the design, dividing the system into different functional areas to achieve the replaceability and controllability of components, thereby giving it broader transformation and clinical application prospects.

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