overview
The APHiGO project consists of three major engineered modules: First, the early detection system employs engineered Bacillus subtilis as a biosensor, enabling it to detect aphid honeydew and convert it into the volatile signal methyl salicylate (MeSA). This system is coupled with an intelligent sensor network for real-time monitoring and early warning through a WeChat mini-program, achieving early detection and monitoring of aphid infestation during the latent period. Secondly, the long-term prevention system utilizes a trunk injection strategy, where iteratively optimized RNAi molecules target key aphid genes. These RNAi molecules are delivered via MS2 viral-like particles fused with penetrating peptides and targeting peptides to ensure efficient entry into aphid intestinal cells, significantly enhancing both control efficacy and stability. Finally, for aphid outbreaks, the rapid treatment system uses engineered Metarhizium fungi to deliver a "dual strike"—combining fungal infection with continuous expression of RNAi molecules. A light-controlled safety switch is integrated to ensure environmental containment of the strain and effectively prevent its spread. Additionally, the project optimizes an in vitro transcription system, ensuring large-scale production of high-purity dsRNA.
In our project, we follow the DBTL (Design-Build-Test-Learn) principle, iteratively optimizing each module. This forms the core of our engineered approach. During the design phase, we rely on biological knowledge, computational models, communication with experts, and experimental results. Each cycle of building, testing, and learning directly feeds back into a new, more targeted design, creating a spiral of continuous optimization. It is through this ongoing DBTL cycle that we have successfully transformed an initial concept into the highly efficient and safe APHiGO.
RNAi Silence module
In the RNAi Silence module, we have designed a total of 4 generations of RNAi molecules. Below, you can read in detail about our Engineering process.
Please click the collapsible boxes below to view our detailed DBTL cycle process
RNAi V1: Single-target dsRNA
▼Design
RNAi V1: The brown citrus aphid (Toxoptera citricida) poses a severe threat to the citrus industry. During in-depth discussions with our principal investigator, Professor Mo, at the early stage of the project, we learned that RNA-based pesticides, as an emerging green pest control technology, combine high efficiency with environmental safety—making them an ideal approach for the precise management of brown citrus aphids. Consequently, we established the use of RNA interference (RNAi) technology as the core strategy for pest control.
Through cross-screening across multiple databases and performing sequence conservation analysis of key genes using the MEGA software, we ultimately identified three genes essential to the growth and development of Toxoptera citricida: chitin synthase (CHS), cytochrome P450 (CYP450), and cuticle protein CP19 (CP). Considering that double-stranded RNA (dsRNA) represents the most mature and widely applied form of RNAi technology in both research and commercial RNA-based pesticides, we constructed three single-target dsRNA molecules in the RNAi V1 phase—dsCHS, dsCYP450, and dsCP—each designed to specifically silence one of the three target genes.
To meet the demand for large-scale dsRNA production during the laboratory phase, we employed a cell-free production system. Plasmids containing the target sequences were first introduced into Escherichia coli for amplification. The extracted plasmids then served as templates for in vitro transcription to synthesize the required dsRNA molecules.
For efficacy evaluation, we adopted a plant-mediated RNAi approach, which most closely simulates the natural oral ingestion of dsRNA and the subsequent gene silencing process in insects. This method allows for a scientifically rigorous assessment of the lethality and biological function of our designed RNAi molecules under conditions approximating real-world application.
Build
During the Build stage, we successfully transformed the constructed recombinant plasmid into E. coli for amplification. After large-scale plasmid extraction, linearized DNA fragments were obtained through double digestion, which served as the standard template for in vitro transcription. After digestion, we used a bidirectional promoter cell-free production system to produce the target dsRNA molecules. Through quantitative analysis of the products using Nanodrop and qualitative analysis of the products using agarose gel electrophoresis (Fig 1), we finally obtained the target single-target dsRNA molecules.
Fig 1. Agarose gel electrophoresis verification of dsCHS transcription products. Lanes 1-6 dsCHS: 300 bp. Marker (left): TAKARA DL500 DNA Marker; Marker (right): TAKARA DL1000 DNA Marker.
To evaluate the gene-silencing efficiency of the RNAi molecules within aphids, we designed and synthesized multiple pairs of qPCR primers targeting the three genes of interest (CHS, CYP450, and CP19) as well as the internal reference gene EF1α. Using conventional PCR and electrophoretic analysis (Fig. 2), we preliminarily assessed the amplification efficiency and product size of each primer pair. Based on these results, TCiCP3, TCiCHS-9, and TCiCYP-14 were selected as the optimal detection primers for CP19, CHS, and CYP450, respectively. Correspondingly, TCiEF1α-2, TCiEF1α-14, and TCiEF1α-5 were chosen as internal reference primers for subsequent qRT-PCR experiments.
Fig 2. qPCR primer validation result. (a) shows the agarose gel electrophoresis results of conventional PCR for the positive reactions of primers CHS1–8 and their negative control (NC); (b) shows the electrophoresis results for the positive reactions of primers sets CHS9–12 and CP1–4, along with their negative control (NC); (c) shows the results for the positive reactions of primer sets CP5–7 and CYP450 1–5, along with their negative control (NC); and (d) shows the results for the positive reactions of primers sets CYP450 8–13 and their negative control (NC).
Test
For the experimental data, we corrected the mortality data according to the Abbott formula. Finally, verification showed that at 120 hours, the corrected mortality rate of aphids in the 400 ng/μl dsCHS treatment group was 11.22%; while that in the 800 ng/μl dsCHS treatment group was 20.48%. The corrected mortality rate of aphids in the 800 ng/μl dsCP treatment group reached 30%, and that in the dsCYP450 treatment group further increased to 31.4% (Fig. 3).
To further verify the molecular-level effects of RNAi, we extracted total RNA from aphids and performed qRT-PCR analysis. The results showed that the 800 ng/μL dsCHS, dsCP, and dsCYP450 treatment groups decreased the expression levels of their corresponding target genes by 27%, 43%, and 30%, respectively (Fig. 4), confirming the effective silencing ability of the constructed RNAi molecules in vivo.
Lethality efficiency curve. (a): Cumulative mortality rates of Toxoptera citricida (brown citrus aphids) treated with 400 ng/μl dsCHS and 800 ng/μl dsCHS, compared with the control group, at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h,72h,84h,96h,108h and 120 h post-treatment. (b): Cumulative mortality rates of Toxoptera citricida (brown citrus aphids) treated with 800 ng/μl dsCHS, 800 ng/μl dsCYP450, and 800 ng/μl dsCP, compared with the control group, at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h,72h,84h,96h,108h and 120 h post-treatment.
Effects of dsCHS, dsCYP450, dsCP RNA interference (RNAi) on CHS, CYP450, CP19(CP) expression level. The relative expression levels of CHS, CYP450, and CP19(CP) in Toxoptera citricida after treatment with 800 ng/μl dsCHS, dsCYP450, and dsCP for 120 h, respectively, compared to the control. Error bars represent the standard deviation (SD) derived from at least three biological replicates. All datasets were statistically analyzed using a Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Learn
Based on the experimental results of the first-generation single-target dsRNA, we found that although RNAi molecules targeting CHS, CYP450, and CP19 could all cause aphid death and significantly reduce the expression of target genes, there were differences in silencing efficiency among different targets, and the overall lethality level still did not reach the ideal control effect.
While designing the first-generation dsRNA, we read literature and communicated extensively. After discussing with Dr. Chen Ruoyu, we introduced the strategy of fusing multi-target dsRNA. By simultaneously silencing multiple key genes, a synergistic lethal effect is produced, and the design of the second-generation molecules was initiated.
In addition, during the cell-free production process, we found that the bidirectional promoter-based transcription system tended to generate non-specific bands exceeding the expected length, which affected the purity of the dsRNA products. To address this issue, we optimized the template design and upgraded the in vitro transcription workflow—details of which are presented in our Cell-Free Synthesis Module.
RNAi V2: Fusion multi-target dsRNA
▼Design
The experimental results of the first-generation single-target dsRNA molecules (dsCHS, dsCYP450, and dsCP) confirmed the effectiveness of the RNAi pathway but also revealed its limitations. Specifically, the insecticidal efficiency of single-gene targeting reached a bottleneck, and literature reports suggest that prolonged use of single-target dsRNA molecules may lead to the development of pest tolerance. To overcome these challenges, we introduced a fusion multi-target dsRNA strategy in the second-generation design.
In the design process, we first developed a statistical algorithm to systematically evaluate the binding efficiency of different targets and nucleotide sites across multiple RNAi prediction databases. Guided by both the program's analytical results and literature recommendations regarding effective interference lengths (~60 bp), we ultimately selected short, high-efficiency dsRNA fragments of 60–100 bp as core modules. Based on this, we constructed dual-fusion dsRNA (dsF2) molecules targeting CHS and CYP450, as well as triple-fusion dsRNA (dsF3) molecules capable of simultaneously targeting CHS, CYP450, and CP19, through direct fusion of the optimized short dsRNA segments.
To assess its biosafety, we aligned the designed fused dsRNA sequence with the transcriptome database of non-target organisms to ensure that its off-target risk is controllable.
Build
During the construction phase, the plasmids pUC57_dsF2 and pUC57_dsF3, each containing the target gene fragments, were transformed into Escherichia coli for amplification. The plasmids were then extracted and digested with restriction enzymes to obtain linearized DNA templates. Using these templates for in vitro transcription, we successfully synthesized dsF2 and dsF3 dsRNA products. The resulting molecules were quantitatively analyzed using a NanoDrop spectrophotometer and qualitatively verified by agarose gel electrophoresis, confirming that the dsRNA products matched the expected lengths (Fig. 5).
Fig 5. Agarose gel electrophoresis verification of dsF2 and dsF3 transcription products. (a)dsF2: 402bp, marker(left):sangon 1000bp marker; marker(right):BBI DNA Marker A (25~500 bp); (b)dsF3: 391bp, marker:sangon 1000bp marker.
Test
During the experimental validation phase, we employed a plant-mediated RNAi approach to evaluate the silencing efficiency and aphid mortality induced by the fusion multi-target dsRNA molecules. RNase free water treatment was used as the negative control, while the experimental groups consisted of 800 ng/μL dsF2 and 800 ng/μL dsF3.
The results showed that the dsF2 treatment group achieved a corrected mortality rate of 40.8% at 120 hours, whereas the dsF3 treatment group exhibited a stronger insecticidal effect, with the corrected mortality rate further increasing to 44.9% (Fig. 6).
At the molecular level, dsF2 treatment reduced the expression of the CYP450 gene by 31.5%. In contrast, dsF3 treatment simultaneously suppressed the expression of CHS, CYP450, and CP19 genes, with expression levels decreased by 35.3%, 20%, and 45%, respectively (Fig. 6). These findings provide strong evidence that the fusion multi-target dsRNA molecules can effectively achieve synergistic gene silencing in vivo.
Fig 6. Lethality efficiency curve & effects of dsCHS, dsCYP450, dsCP RNA interference (RNAi) on CHS, CYP450,CP19(CP) expression level. (a): Cumulative mortality rates of brown citrus aphids treated with 800 ng/μl dual-fusion dsRNA (dsF2) or triple-fusion dsRNA (dsF3) at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h, compared with the control. (b): Relative expression levels of CHS and CYP450 in brown citrus aphids treated with 800 ng/μl dsF2 for 120 h, compared with the control.Error bars represent the standard deviation (SD) derived from at least three biological replicates. All datasets were statistically analyzed using a Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (c): Relative expression levels of CHS, CYP450, and CP19(CP) in brown citrus aphids treated with 800 ng/μl dsF3 for 120 h, compared with the control.Error bars represent the standard deviation (SD) derived from at least three biological replicates. All datasets were statistically analyzed using a Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Learn
Based on the results of the second-generation fusion dsRNA experiments, we found that the multi-target synergistic strategy effectively enhanced insecticidal efficacy. However, through further discussions with field experts, we recognized that long-chain dsRNA molecules are prone to degradation by RNases in complex environments and may pose potential off-target risks, both of which limit their practical applications.
To address these challenges, in our third-generation design, we introduced a short hairpin RNA (shRNA) structure to improve molecular stability.
Meanwhile, following in-depth discussions with nanocarrier specialist Dr. Xuedong Liu, we explored an additional stabilization approach—encapsulation of RNA molecules using MS2 virus-like particles (MS2 VLPs)—to further enhance delivery efficiency and environmental stability. Details of this work can be found in the MS2 VLP Module.
During the experimental process, we also noted that existing dsRNA design tools lack functional support for constructing complex multi-target fusion sequences. To overcome this limitation, we upgraded our initially developed statistical algorithm into a comprehensive design platform named APHiGEM, enabling efficient and rational design of complex RNAi molecules (see Software page for details).
RNAi V3: Multi-tandem shRNA(tri-shRNA)
▼Design
In the third-generation RNAi design, inspired by the results of the previous two generations, we developed a multi-tandem short hairpin RNA (tri-shRNA) architecture. This design integrates multiple shRNA units—each specifically targeting one of the key genes CHS, CYP450, and CP19—into a single tri-shRNA construct through tandem linkage using the classical hairpin loop sequence TTCAAGAGA.
This structural design effectively minimizes redundant sequences and reduces off-target risks, while the stem–loop configuration enhances the overall stability of the RNAi molecule. Furthermore, RNAfold secondary structure simulations confirmed that each shRNA unit within the tandem system maintains structural independence, supporting the rationality and feasibility of the tri-shRNA design.
Build
We first performed structural simulations of the designed tri-shRNA using RNAfold, and the predicted secondary structure results (Fig. 7) confirmed the rationality of the design. Subsequently, the constructed plasmids were transformed into E. coli for amplification, and the transcribed products were synthesized using a cell-free expression system. The resulting tri-shRNA molecules were successfully obtained and verified to match the expected length (Fig. 8).
Fig 7. Secondary structure prediction of tri-shRNA
Fig 8. Agarose gel electrophoresis verification of tri-shRNA in vitro transcription products. Marker ( left ) : DL5000 DNA Marker, 1 : negative control, 2、3、4 : tri-shRNA transcription product213bp Marker ( right ) : D2000 DNA Marker.
Test
During the experimental validation phase, we employed a plant-mediated RNAi approach to assess the mortality induced by the tri-shRNA construct. RNase free water treatment served as the negative control, while the experimental group was treated with 800 ng/μL tri-shRNA. The results showed that the tri-shRNA treatment group achieved a corrected mortality rate of 49.5% at 120 hours (Fig. 9).
Fig 9. Lethality efficiency curve Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h after treatment with 800 ng/μl tri-shRNA and in the control group.
Learn
Based on the experimental results of the tri-shRNA, we observed an improvement in both synergistic silencing efficiency and molecular stability compared with the previous generations. However, through discussions with the HUBU-China team, we realized that different species exhibit distinct RNAi-processing mechanisms, and that the conventional hairpin loop structures may have limited processing efficiency within aphid cells.
To achieve more efficient gene silencing, in our fourth-generation design, we incorporated the endogenous microRNA (miRNA) processing pathway of aphids. Specifically, we constructed artificial microRNA (amiRNA) based on the native aphid miRNA scaffold, aiming to enhance both the processing efficiency and silencing specificity of RNAi molecules within the target species.
RNAi V4: Tandem amiRNA(bi-amiRNA)
▼Design
Building upon the advantages of high molecular stability and low off-target risk observed in shRNA constructs, we introduced an artificial microRNA (amiRNA) design in our fourth-generation RNAi system. By precisely embedding specific interference sequences into the endogenous aphid pre-miRNA scaffold, this strategy effectively harnesses the host's natural RNAi processing machinery to achieve efficient gene silencing.
To further enhance synergistic interference, we constructed a dual-tandem amiRNA structure(bi-amiRNA) capable of simultaneously targeting the two key genes, CHS and CYP450. This design continues our multi-target regulatory strategy while enabling more efficient gene silencing within the pest.
Build
During the Build phase, we first screened the miRBase database and selected two endogenous aphid pre-miRNAs, api-mir-71 and api-mir-3017a, as biomimetic scaffolds (Fig. 10). The validated high-efficiency siRNA sequences targeting CHS and CYP450 were then substituted into the mature miRNA regions of these scaffolds. The two units were subsequently linked using an AGGCAT linker to construct a dual-tandem bi-amiRNA. Secondary structure analysis using RNAfold confirmed that each amiRNA unit maintained structural independence within the tandem configuration (Fig. 11).
Fig 10. The screened aphid miRNA scaffolds. (a) api-mir-71 scaffold (b) api-mir-3017a scaffold
Fig 11. Secondary Structure Prediction of the bi-amiRNA
The recombinant plasmids were transformed into Escherichia coli for amplification, and the bi-amiRNA molecules were subsequently synthesized using an in vitro transcription system. The resulting products exhibited complete structural integrity and the expected molecular length. Their purity and integrity were further verified through agarose gel electrophoresis (Fig. 12).
Fig 12. Agarose gel electrophoresis analysis of in vitro transcribed tri-shRNA.Marker ( left ) : DL5000 DNA Marker, 1 : negative control, 2、3、4 : tri-shRNA transcription product——157bp, Marker ( right ) : D2000 DNA Marker
Test
During the experimental validation phase, we employed a plant-mediated RNAi approach to evaluate the insecticidal efficacy of the bi-amiRNA construct. RNase free water treatment served as the negative control, while the experimental group was treated with 800 ng/μL bi-amiRNA. The results showed that the bi-amiRNA treatment group achieved a corrected mortality rate of 60.2% at 120 hours (Fig. 13). Among all four generations of RNAi molecules, bi-amiRNA demonstrated the highest insecticidal efficiency, representing the most effective construct in our RNAi series.
Fig 13. Lethality efficiency curve Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h after treatment with 800 ng/μL bi-amiRNA and inthe control group.
Learn
Based on the experimental results of the fourth-generation bi-amiRNA, we successfully validated the advantage of the biomimetic design in enhancing RNAi efficiency. Both the lethal effect and response speed of bi-amiRNA were significantly superior to those of the previous three RNAi systems (Fig. 14).
Fig 14. Cumulative mortality rates of Toxoptera citricida (brown citrus aphids) treated with 400 ng/μl dsCHS, 800 ng/μl dsCHS, dsCYP450, dsCP, dsF2, dsF3, tri-shRNA, and bi-amiRNA, compared with the control group, measured at 6, 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 hours post-treatment.
However, in field applications, RNAi formulations still face challenges related to persistence and durability. To achieve more effective pest control under field conditions, we explored the integration of biological delivery systems with RNAi technology by employing engineered Metarhizium anisopliae as a living carrier. This approach establishes a dual insecticidal mechanism that combines fungal infection with RNAi-mediated gene silencing. Such a fusion strategy not only leverages the fungus's ability to colonize crop surfaces persistently, but also enables specific silencing of target genes, thereby offering a novel and sustainable pathway for the green management of citrus aphids.
MS2 VLP module
From the RNAi V2 experiments, we recognized that enhancing the environmental stability of RNAi molecules is crucial for their practical application. To address this, we introduced MS2 virus-like particles (MS2 VLPs) as a protective delivery carrier. Leveraging their self-assembly properties, MS2 VLPs can efficiently encapsulate RNAi molecules containing the pac site, thereby preserving the structural integrity and functional activity of RNAi constructs under complex environmental conditions.
1st-gen VLP
Design
To enhance the stability of RNAi molecules in complex environmental conditions, we constructed an in vivo self-assembling delivery system based on MS2 virus-like particles (VLPs). This system exploits the specific recognition between the MS2 coat protein and the pac site on RNA molecules. By co-expressing the coat protein and pac site–containing RNAi molecules in Escherichia coli, the two components spontaneously assemble intracellularly to form structurally complete VLP–RNAi complexes.
This design not only provides effective protection against environmental nuclease degradation but also features nanoscale particle size, which facilitates efficient delivery and uptake within biological systems. Collectively, this MS2 VLP–based strategy offers a robust protective platform for advancing the practical application of RNAi technology.
Build
We successfully constructed the recombinant plasmid pACYCDuet_1-CP-HisTag-CP-amiRNA, which contains the MS2 coat protein dimer sequence, a His-tag, and the target amiRNA expression cassette. A pac site was fused to one end of the target amiRNA to facilitate specific encapsulation. After sequence verification confirmed the correct plasmid construction, the recombinant vector was transformed into Escherichia coli BL21(DE3) for expression.
To verify the successful encapsulation of the target RNA by VLPs, we performed a nuclease protection assay. Following gradient nuclease treatment, the RNA within the encapsulated group remained intact, whereas free RNA was completely degraded. Coomassie Brilliant Blue staining simultaneously confirmed the stable presence of coat proteins, demonstrating that the assembled VLP complexes effectively protected the RNA payload.
To further assess the morphology of the virus-like particles, we conducted transmission electron microscopy (TEM) analysis. The purified samples displayed uniform spherical particles with well-defined boundaries under negative staining conditions (Fig. 17A, B), confirming the successful assembly of complete amiRNA-loaded MS2 VLPs (Fig. 17).
Fig 17. Transmission Electron Microscopy (TEM) images of purified histidine-tagged MS2 phage-like particles (histidine-tagged VLPs). The scale bar represents 100 nanometers.
Test
We evaluated the protective performance of the VLPs through a nuclease resistance assay. Naked RNA and VLP-encapsulated RNA were separately treated with 100 U/mL Benzonase Nuclease at 37 °C. The electrophoresis results showed that naked RNA was completely degraded within 10 minutes, whereas the RNA encapsulated within VLPs remained intact even after 30 minutes of treatment (Fig. 18). These findings demonstrate that the VLPs provide an effective nuclease barrier for RNA molecules, significantly enhancing their stability under complex environmental conditions.
Fig 18. Agarose gel electrophoresis demonstrating the nuclease resistance of amiRNA encapsulated in virus-like particles (VLPs). Lanes 1–4: 50 µM VLPs treated with 100 U/mL Benzonase nuclease for 0, 10, 20, and 30 minutes, respectively; Lanes 6–9: 500 nM naked amiRNA treated under the same conditions for the corresponding durations. Results show that VLPs effectively protect amiRNA from degradation under high nuclease conditions, whereas naked amiRNA is completely degraded within 10 minutes, confirming the significant protective effect of VLPs on RNA.
Learn
The first-generation MS2 VLP system successfully achieved efficient in vivo self-assembly encapsulation of RNAi molecules, and nuclease protection assays confirmed its ability to effectively maintain RNA stability under complex environmental conditions. However, transmission electron microscopy (TEM) and gel mobility analyses revealed that the assembled VLP complexes exhibited a relatively large particle size (approximately 27 nm), which may limit their tissue penetration and cellular delivery efficiency in biological systems.
To overcome this bottleneck, the next-generation system will incorporate a functional peptide modification strategy: by fusing the TAT cell-penetrating peptide to enhance intracellular delivery capability, and introducing the GBP3.1 gut-targeting peptide to improve VLP accumulation efficiency in the aphid midgut tissues. This design aims to retain the excellent protective properties of the original VLPs while further strengthening their targeting specificity and cellular internalization efficiency.
2nd-gen VLP
Design
To enhance the targeted delivery efficiency of VLPs within aphids, we integrated a dual-functional peptide modification system into the MS2 capsid protein. Specifically, the fusion of the GBP3.1 gut-targeting peptide enables specific accumulation in the aphid midgut, while incorporation of the TAT cell-penetrating peptide enhances intracellular delivery capacity. Through precise molecular docking and spatial conformation optimization, this design preserves the self-assembly capability of the capsid protein and establishes a second-generation VLP delivery system with both tissue-targeting and transmembrane transport functions.
Build
During the construction phase, we successfully generated three functional validation vectors: pET28a_GBP3.1-eGFP-TAT-HisTag (for evaluating the targeting and penetration effects of the dual-functional peptides), pET28a_eGFP (as a control vector), and pET28a_CP-GBP3.1-CP-TAT-HisTag (for expressing the functionalized capsid protein). These plasmids were individually transformed into Escherichia coli BL21(DE3), and induction expression yielded highly purified target proteins, providing essential materials for subsequent functional verification experiments.
Test
To evaluate the targeting and cell-penetrating capabilities of GBP3.1 and TAT peptides within aphids, we conducted a feeding assay comparing the in vivo distribution of the GBP3.1-EGFP-TAT fusion protein with that of the EGFP control. Live fluorescence imaging (Fig. 19) and immunofluorescence analysis of tissue sections (Fig. 20) revealed markedly enhanced green fluorescence signals in aphids treated with the fusion protein, with fluorescence specifically localized in intestinal and other target tissues. These results demonstrate that GBP3.1 and TAT synergistically mediate efficient translocation of exogenous proteins across the aphid gut barrier and facilitate intracellular delivery, while GBP3.1 additionally confers distinct tissue-targeting specificity.
Fig 19. In vivo fluorescence imaging of aphids.Left: EGFP control group; Right: GBP3.1-EGFP-TAT experimental group. The experimental group shows significant green fluorescence throughout the body, while the control group only exhibits weak autofluorescence from chitin (arrows), demonstrating the effective delivery and retention of EGFP mediated by the dual-functional peptides. Scale bar: 0.5 mm.
Fig 20. Immunofluorescence imaging of aphid intestinal tissue sections.Left: EGFP control group; Right: GBP3.1-EGFP-TAT experimental group. Specific green fluorescence aggregation is observed in the midgut region of the experimental group, indicating the intestinal targeting capability of GBP3.1 and the tissue penetration/diffusion enhancement by TAT. Blue: DAPI nuclear staining. Scale bar: 50 μm.
Learn
In the iterative development of the engineered VLP system, the incorporation of a dual-functional peptide modification strategy significantly enhanced delivery efficiency within aphids through the synergistic action of targeting and membrane-penetrating mechanisms. However, the introduction of functional peptides may partially compromise the self-assembly efficiency of the capsid protein, leading to reduced yield and stability. To address this, future work will focus on systematically evaluating the assembly efficiency and structural stability of the modified VLPs, while optimizing the linker region between the functional peptides and the capsid protein to further improve the reproducibility and robustness of the functionalized VLP system.
Cell-free synthesis module
CFS-I
Design
To meet the demand for large quantities of high-purity dsRNA during multiple iterations of RNAi experiments, we designed and established a scalable production platform based on cell-free synthesis technology. This platform employs a bidirectional T7 promoter system to efficiently generate structurally intact dsRNA molecules through a one-step transcription process, providing a stable and reliable source of RNAi materials for subsequent functional validation.
Build
At the Build stage, the constructed recombinant plasmids — pUC57_CHS, pUC57_dsF2, and pUC57_dsF3 — were transformed into Escherichia coli DH5α competent cells using the heat-shock method. Following ampicillin-resistant colony screening, shake-flask amplification, and plasmid extraction, the linearized DNA templates were obtained through double-enzyme digestion.
Test
During the in vitro transcription process, the purified linearized DNA templates were incubated with T7 RNA polymerase, NTPs, and transcription buffer at 37 °C for 4 hours. Using the bidirectional promoter system, complementary RNA strands were simultaneously transcribed. The reaction products were subsequently treated with DNase I to remove residual DNA templates, yielding high-purity double-stranded RNA (Fig. 21) suitable for direct use in downstream functional assays.
Fig 21. Agarose gel electrophoresis verification of dsF2 and dsF3 transcription products. (a)dsF2:402bp,marker(left):sangon 1000bp marker,marker(right):BBI DNA Marker A (25~500 bp) (b)dsF3:391bp,marker:sangon 1000bp marker
Learn
During the establishment and optimization of the cell-free transcription system, we successfully achieved efficient synthesis of the target dsRNA, providing stable material support for multiple rounds of RNAi iteration experiments. However, product analysis revealed a pronounced read-through phenomenon in transcription systems lacking effective terminators, leading to the generation of elongated, non-target RNA fragments. These byproducts not only reduced the yield of functional dsRNA but also introduced potential off-target risks due to nonspecific sequences. Through systematic literature research, we incorporated a multi-terminator array to construct a hierarchical termination signal system, thereby improving transcription termination efficiency and overall dsRNA yield.
CFS-Ⅱ
Design
To meet the increasing demand for high-yield and high-purity dsRNA production in future RNAi iteration experiments, we introduced a triple-terminator array (T7UUCG–rrnB T1–T7 terminator) while retaining the bidirectional T7 promoter. The cooperative action of these multilayered termination signals effectively reduced transcriptional read-through, thereby improving both the purity and yield of the target dsRNA products. To further refine this strategy, we adopted a modular design approach by introducing restriction enzyme sites between adjacent terminator units, enabling the construction of gradient plasmids containing single (1T), double (2T), and triple (3T) terminator configurations. By comparing the effects of different terminator arrays on transcriptional accuracy and product yield, we established a practical foundation for the precise regulation of cell-free transcription systems.
Build
Based on the pUC57_CHS construct, we introduced a triple-terminator cassette to generate pUC57_CHS-tri-terminator. Following digestion with different restriction enzymes, linearized DNA templates were prepared and verified by agarose gel electrophoresis. The results confirmed the successful acquisition of linear templates within the target band region (Fig. 22).
Fig 22. Agarose gel electrophoresis analysis (a):MW:marker1,2:TAKARA DL1000 DNA marker; marker3: BBI DNA Marker A (25~500 bp) 1:CHS-1T(456bp);2:CHS-2T(594bp)(b)MW:marker(left):TAKARA DL1000 DNA marker; marker(right): BBI DNA Marker A (25~500 bp) 1:CHS-NT(346bp);2:CHS-3T(716bp)
Test
Using the same in vitro transcription system for validation, the results showed that compared with the first-generation system, the introduction of the triple-terminator array significantly reduced non-specific transcriptional bands (Fig. 23). Agarose gel electrophoresis revealed markedly improved product homogeneity, confirming that the multilayer termination signals effectively suppressed transcriptional read-through.
Fig. 23. Agarose gel electrophoresis analysis (a) Agarose gel electrophoresis analysis illustrating the comparison of dsRNA purity; (b) Agarose gel electrophoresis analysis comparing transcriptional yield. MW: molecular weight marker; NT-CHS: CHS dsRNA transcribed from a template lacking terminators; 1T-CHS: CHS dsRNA transcribed from a template containing a single T7UUCG terminator; 2T-CHS: CHS dsRNA transcribed from a dual-terminator template containing tandem T7UUCG and rrnB T1 terminators; 3T-CHS: CHS dsRNA transcribed from a triple-terminator template containing tandem T7UUCG, rrnB T1, and T7 terminators.
Meanwhile, RNA samples were diluted with nuclease-free water at 1×, 5×, and 10× concentrations and quantified using a Nanodrop spectrophotometer. The results showed that dsCHS transcribed from the triple-terminator template exhibited a significantly higher RNA concentration compared with those transcribed from other templates (Fig. 24).
Fig. 24. Significance analysis of RNA concentrations for transcription products (a) Significance analysis of RNA concentrations for NT-, 1T-, 2T-, and 3T-template transcription products at 1× dilution; (b) significance analysis at 5× dilution; (c) significance analysis at 10× dilution; (d) linear regression curves describing the relationship between dilution ratio and RNA concentration. Regression equations: NT-CHS：YNT=6317*X-29.37,R2=0.9967；1T-CHS：Y1T=9538*X+164.8,R2=0.9997；2T-CHS： Y2T=9604*X-100.3,R2=0.9969；3T-CHS：Y3T=11006*X+240.3,R2=0.9971。ns：p>0.05,*：p <0.05,**：p <0.01,***：p<0.001,****：p<0.0001
Learn
Through a systematic evaluation of different terminator arrays in the cell-free transcription system, we confirmed that the triple-terminator array (3T) confers a significant advantage in improving both the yield and purity of dsRNA. Linear regression analysis further demonstrated that the 3T template maintained the highest concentration stability across all dilution gradients (R² = 0.9971), indicating superior transcriptional efficiency and controllability. This optimization not only provides a reliable approach for large-scale production of high-purity dsRNA but also establishes a solid technical foundation for the future development of more complex RNA molecules.
References
Toé, H. K., Zongo, S., Guelbeogo, M. W., Kamgang, B., Viana, M., Tapsoba, M., ... & Sagnon, N. F. (2022). Multiple insecticide resistance and first evidence of V410L kdr mutation in Aedes (Stegomyia) aegypti (Linnaeus) from Burkina Faso. Medical and veterinary entomology, 36(3), 309-319.
Shang, Q., Lu, H., Yang, M., Wu, Y., & Chen, Q. (2024). The Advancement and Prospects of the Tree Trunk Injection Technique in the Prevention and Control of Diseases and Pests. Agriculture, 14(1), 107.
Wangpaiboon, K., Klaewkla, M., Charoenwongpaiboon, T., Vongkusolkit, N., Panpetch, P., Kuttiyawong, K., ... & Pichyangkura, R. (2022). Synergistic enzyme cocktail between levansucrase and inulosucrase for superb levan-type fructooligosaccharide synthesis. Enzyme and Microbial Technology, 154, 109960.
Zhu KY, Palli SR. Mechanisms, Applications, and Challenges of Insect RNA Interference. Annu Rev Entomol. 2020 Jan 7;65:293-311.
Yang Wang, Xianpeng Yang, Zhaoxing Chen, Jin Zhang, Kai Si, Rangwei Xu, Yizhong He, Feng Zhu, Yunjiang Cheng, Function and transcriptional regulation of CsKCS20 in the elongation of very-long-chain fatty acids and wax biosynthesis in Citrus sinensis flavedo, Horticulture Research, Volume 9, 2022, uhab027.
Mahanta, D. K., Komal, J., Bhoi, T. K., Samal, I., Dash, S., & Jangra, S. (2025). RNA interference (RNAi) for insect pest management: understanding mechanisms, strategies, challenges and future prospects. Biologia Futura, 1-13.
Shang, F., Xiong, Y., Xia, W. K., Wei, D. D., Wei, D., & Wang, J. J. (2016). Identification, characterization and functional analysis of a chitin synthase gene in the brown citrus aphid, Toxoptera citricida (Hemiptera, Aphididae). Insect Molecular Biology, 25(4), 422-430.
Jing, T. X., Tan, Y., Ding, B. Y., Dou, W., Wei, D. D., & Wang, J. J. (2018). NADPH–cytochrome P450 reductase mediates the resistance of Aphis (Toxoptera) citricidus (Kirkaldy) to abamectin. Frontiers in Physiology, 9, 986.
Shang Feng, S. F., Ding BiYue, D. B., Ye Chao, Y. C., Yang Li, Y. L., Chang TengYu, C. T., Xie JiaQin, X. J., ... & Wang JinJun, W. J. (2020). Evaluation of a cuticle protein gene as a potential RNAi target in aphids.
Mou, X., Yuan, G. R., Jiang, H. B., Liu, Z., & Wang, J. J. (2017). Functional characterization of two acetylcholinesterase genes in the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), using heterologous expression and RNA interference. Pesticide Biochemistry and Physiology, 138, 76-83.
Wang, Z. G., Qin, C. Y., Chen, Y., Yu, X. Y., Chen, R. Y., Niu, J., & Wang, J. J. (2024). Fusion dsRNA designs incorporating multiple target sequences can enhance the aphid control capacity of an RNAi‐based strategy. Pest Management Science, 80(6), 2689-2697.
Rajput, I.A., Ahmed, A.M., Khan, K.A. et al. Multitrophic interactions between coccinellids and their parasitoids in natural and managed plant systems: host plant and aphid prey species matter. Int J Trop Insect Sci 43, 2011–2020 (2023).
Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol Biol. 2010;629:141-58.
Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol Biol. 2010;629:141-58.
Jiang, C., Fu, J., Li, F., Xia, K., Li, S., Chang, L., ... & Zhang, J. (2024). Plastid engineering with an efficient RNAi delivery system based on bacteriophage MS2 virus-like particles enhances plant resistance to cotton bollworm. Molecular Plant, 17(7), 987-989.
Dang, M., Wu, L. J., Zhang, S. R., Zhu, J. R., Hu, Y. Z., Yang, C. X., & Zhang, X. Y. (2023). MS2 Virus-like particles as a Versatile peptide presentation platform: insights into the deterministic abilities for accommodating heterologous peptide lengths. ACS Synthetic Biology, 12(12), 3704-3715.
Juliana da Rosa, Américo José Carvalho Viana, Fernando Rafael Alves Ferreira, Alessandra Koltun, Liliane Marcia Mertz-Henning, Silvana Regina Rockenbach Marin, Elibio Leopoldo Rech, Alexandre Lima Nepomuceno, Optimizing dsRNA engineering strategies and production in E. coli HT115 (DE3), Journal of Industrial Microbiology and Biotechnology, Volume 51, 2024, kuae028.
