Improvement

The introduction of our experiment

Our improved experiment is based upon 2022 Jinan Foreign Language School team(https://parts.igem.org/Part:BBa_K4167666) and 2024 cjuh-jlu20-china team (https://2024.igem.wiki/cjuh-jlu-china/description) , which all used technology for disease detection based on RNA secondary structure. The project had applied Toehold Switch -- a class of riboregulators that can be activated when interacting with a specific trigger RNA, a single loop nucleic acid detection system to detect a significantly elevated microRNA in MDD, miR-34a-5p. sLIRA-miR34A-5p is the sequence of the single-loop toehold nucleic acid detection system.

Figure 1.Sequence components and secondary structure of a toehold switch

Improved part：

This year, our YiYe-China team is employing the toehold switch for breast cancer diagnosis. However, one major limitation of conventional toehold switches lies in their structural rigidity and susceptibility to misfolding or false activation under varying in vitro conditions. In order to enhance detection efficiency, we aim to make further efforts in the following aspects to improve the completeness of the project. We propose an improved toehold architecture featuring a Loop-Initiated Isothermal RNA Activator (LIRA) and the integration into a cell-free transcription-translation system.

The Loop-Initiated Isothermal RNA Activator (LIRA) is a novel RNA detection technology based on RNA secondary structure. The single-arm structure of LIRA consists of a loop and complementary paired stem, with its recognition domain divided into two parts located within the stem (b*) and the loop (a*). LIRA hides the ribosome binding site (RBS) sequence and the start codon AUG necessary for RNA translation within the stem-loop structure. When the target miRNA is present, it binds to the LIRA recognition domain, disrupting its original structure and exposing the RBS and AUG, thereby initiating the translation of downstream reporter genes (Figure 2).

Figure 2. Sequence components and secondary structure of LIRA

We employed a two-input AND gate, which is a single toehold switch activated by two trigger

RNAs—miR-34a-5P and miR-93-3p.

miR‑34a‑5p: a overexpression microRNA in breast cancer

Upregulation of miR‑34a makes it a reliable biomarker for breast cancer. Research shows that compared with healthy people, miR-34a-5p significantly increases in circulating blood of breast cancer patients.Small interfering RNA (siRNA)-mediated knockdown of miR-34a significantly inhibits the proliferation of the breast cancer cell line MCF-7[1]. This indicates that overexpression of miR-34a may be an acquired characteristic during carcinogenesis and supports the cell proliferation of breast tumors.In a study of Egyptian patients with locally advanced breast cancer, circulating miR‑34a was significantly higher at diagnosis compared to healthy individuals[2].These findings highlight miR‑34a’s potential as a non-invasive, blood-based biomarker for tracking therapeutic efficacy and stratifying patient prognosis. Since circulating miRNAs are stable and easily detected in plasma, miR‑34a-particularly the active 5p strand—offers a valuable tool for real-time, minimally invasive monitoring of breast cancer progression and treatment outcomes.

hsa‑miR‑93‑3p: an oncogenic strand associated with TNBC progression

 Upregulated in TNBC and linked to poor survival:
Analysis of 1,299 breast tumors from the METABRIC cohort revealed that hsa‑miR‑93‑3p is among ten miRNAs significantly overexpressed in TNBC (n = 204) versus non‑TNBC (n = 1,095), and its high expression correlates exclusively with reduced overall survival in TNBC patients[3].

 Promotes chemoresistance via Wnt/β‑catenin activation:
Functional assays demonstrated that hsa‑miR‑93‑3p confers resistance to cisplatin by downregulating the Wnt antagonist SFRP1, thereby activating the Wnt/β‑catenin pathway, resulting in drug resistance and tumor progression.

 Potential non‑invasive biomarker:
Circulating levels of hsa‑miR‑93‑3p have been proposed as a potential non-invasive biomarker, as it has been proven to be correlated with tumor growth. It can both serve as an indicator of cancer in primary stages and be used to verify the effectiveness of chemotherapy (Li et al., 2017)[3].Quantitative real-time PCR (qPCR) results demonstrated that the expression of miR-93-3p is significantly increased in the MCF-7 cell line as well as in breast cancer tissues[4].

The input for the corresponding toehold switch consists of two input RNA molecules that are partially complementary to each other. This design enhances detection specificity by requiring the simultaneous presence of two target RNAs for activation, reducing false positives and background leakage. It is ideal for precise diagnosis of complex diseases and offers logical control, modular design, and system scalability.

We innovatively embedded our LIRA into a cell-free transcription-translation platform assembled by mixing the two PURExpress solutions (which contain cell-free reaction components) with the substrate, DNA template, and RNase inhibitor. These reactions are then applied to the discs. It provides several advantages. The cell-free setup avoids live-cell handling, ensuring biosafety and enabling easy deployment at point-of-care or in the field. Transcription and translation begin immediately upon sample addition, enabling rapid response. Furthermore, the system is tunable, allowing precise adjustment of component concentrations to optimize sensitivity. It also supports freeze-drying: all reagents can be pre-loaded on paper-based disks, stored at room temperature, and reactivated simply by adding water or biological fluids.

Once the target miRNAs (miR-34a-5P and miR-93-3p) are added, the DLT structure unfolds, exposing the RBS and start codon to initiate translation of EGFP. Fluorescence is observable within 45 minutes.

Result

To improve the detection module of our breast cancer project, we designed and constructed three types of plasmids. The first plasmid contains the reverse complementary sequence of miR-34a-5p, functioning as a single “lock.” The second plasmid carries reverse complementary sequences of both miR-34a-5p and miR-93-3p, forming two “locks.” The third plasmid contains the actual miR-34a-5p and miR-93-3p sequences, serving as detection targets. All synthetic sequences were inserted downstream of the T7 promoter to ensure transcriptional expression. As shown in Figure 1, the plasmid maps validate the structural design of each construct. To verify successful transformation, we plated the bacteria on LB agar plates supplemented with selective antibiotics. Plasmids A and B carry a kanamycin resistance gene, while plasmid C contains a streptomycin resistance gene. Only bacteria that successfully received the plasmid can survive and form colonies under the corresponding antibiotic pressure. The presence of dense colonies on all plates indicates effective uptake of the plasmids. Furthermore, sequencing results in Figure 2 suggest that the inserted sequences are correct, showing no obvious base mismatches or unexpected alterations. Together, these results demonstrate the successful construction of all three plasmids and complete the first phase of our improved detection system.

Figure 1. Construction of three plasmids.

A.pCOLAD-sLIRA-miR34A-5p-EGFP. B.pCOLAD-dLIRA-miR-34a-5p/93-3p-EGFP. C.pCDFD-miR-93-3p/miR-34a-5p

 

Figure 2. The three plasmids sequencing

 

 

Due to the different antibiotic resistance genes(KanR and SmR)carried by overexpression plasmids and detection report plasmids, in order to validate the normal function of our dual loop detection system in vitro, we grouped three plasmids and co transformed them into BL21 competent state. We screened five recombinant BL21 transformed strains (co transformed groups were subjected to two antibiotic screenings), as shown in Figure 3, which are:

1.pCOLAD-sLIRA-EGFP

2.pCOLAD-dLIRA-EGFP

3.pCDFDuet-1-miR-93-3p/miR-34a-5p

4.pCOLAD-sLIRA-EGFP+pCDFDuet-1-miR-93-3p/miR-34a-5p

5.pCOLAD-dLIRA-EGFP+pCDFDuet-1-miR-93-3p/miR-34a-5p

Figure 3. Recombinant BL21 positive colony

 

Next, we conducted in vitro bacterial induction experiments to demonstrate the normal function of our detection system. The recombinant bacteria were inoculated into LB broth containing streptomycin and/or kanamycin resistance and cultured overnight (14-16 hours). Then, 100 μ L of bacterial solution was added to 10ml of LB medium containing fresh streptomycin and kanamycin resistance, and the bacterial OD600 value was controlled at 0.4-0.6. Then add 1mM IPTG and induce overnight at 16 degrees. Take 100ul of bacterial solution and add it to a 96 well enzyme-linked immunosorbent assay (ELISA) plate. Observe the fluorescence under a fluorescence microscope or centrifuge to observe the fluorescence of the bacterial strain precipitate. Take another portion of bacterial solution into the 96 well ELISA plate and use a fluorescence ELISA reader to detect the fluorescence value of the sample. As shown in Figure 4, there was no Green fluorescence observed in the 123 bacterial solutions, while the 45 bacterial solutions co transformed showed significant Green fluorescence. This experiment demonstrates that our designed and synthesized dual ring LIRA detection system can function correctly.

Figure 4. Fluorescence of different recombinant BL21 bacterial induction groups

A. Visible color of bacterial liquid.B.Green fluorescence of bacterial liquid centrifugal precipitation.C.Fluorescence values of various bacterial solutions detected by fluorescence spectrophotometer

 

In clinical practice, serum samples can be collected from healthy individuals and breast cancer patients. The cell - free protein expression system is employed in vitro to mimic the intracellular transcription and translation processes. This allows for the detection of the expression levels of two miRNAs in breast cancer patients, thereby providing assistance in the diagnosis of breast cancer. By reconstituting key components involved in transcription and translation outside the cell, the system enables the synthesis of proteins. Specifically, plasmids which carrying target gene information are transcribed into mRNA and then translated into proteins within this reconstituted environment.Like the in vitro induction experiment of BL21 E. coli, we set up six groups of samples in the cellfree experiment, each of which added the following components：

A. pCOLAD-sLIRA-EGFP groupB. pCOLAD-dLIRA-EGFP groupC. pCDFDuet-1-miR-93-3p/miR-34a-5p groupD. pCOLAD-sLIRA-EGFP、pCDFDuet-1-miR-93-3p/miR-34a-5p groupE. pCOLAD-dLIRA-EGFP、pCDFDuet-1-miR-93-3p/miR-34a-5p groupF. the positive control group

Figure 5. Fluorescence of various groups in cell-free protein expression system

 

Excluding the positive control group F in the six cell free systems, only the DE group was able to transcribe miR-34a-5p or miR-34a-5p/miR-93-3p.So only Group D and Group E exhibit green fluorescence.

In conclusion, we have improved the traditional single loop toehold nucleic acid detection system. Based on the independent detection of miR-34a-5p in blood or tissue samples of breast cancer patients, we have added the detection index of miR-93-3p. The double loop LIRA detection system we designed is more accurate than the single loop detection system, providing a stronger impetus for clinical diagnosis and detection of breast cancer.

Reference:

1.Kassem, N. M., Makar, W. S., Kassem, H. A., Talima, S., Tarek, M., Hesham, H., & El-Desouky, M. A. (2019). Circulating miR-34a and miR-125b as promising non invasive biomarkers in Egyptian locally advanced breast cancer patients. Asian Pacific Journal of Cancer Prevention: APJCP, 20(9), 2749.

2. Li, H.-Y., Liang, J.-L., Kuo, Y.-L., Lee, H.-H., Calkins, M. J., Chang, H.-T., Lin, F.-C., Chen, Y.-C., Hsu, T.-I., & Hsiao, M. (2017). miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer. Breast Cancer Research, 19(1), 133.

3. Li, H. Y., Liang, J. L., Kuo, Y. L., Lee, H. H., Calkins, M. J., Chang, H. T., Lin, F. C., Chen, Y. C., Hsu, T. I., Hsiao, M., Ger, L. P., & Lu, P. J. (2017). miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer. Breast cancer research : BCR, 19(1), 133. 

4. Chu, S., Liu, G., Xia, P., Chen, G., Shi, F., Yi, T., & Zhou, H. (2017). miR-93 and PTEN: Key regulators of doxorubicin-resistance and EMT in breast cancer. Oncology reports, 38(4), 2401–2407.