Latest revision as of 12:11, 28 September 2024

Toehold switch-LacZ

Toehold switch-LacZ is designed to express β-galactosidase triggered by miRNA 34a-5p. It is used to detect the amount of miRNA 34a-5p in samples.

Usage and Biology

Sequence and Features

To construct the standard part, toehold switch-LacZ was amplified and checked the restriction enzyme information, which is shown as follows:

Fig.1 The map of toehold switch-LacZ described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).


Toehold switch-LacZ plasmid was designed to express β-galactosidase controlled by the toehold switch and miRNA 34a-5p. It comprises the antisense sequence of miRNA 34a-5p, RBS, Linker and part sequence of miRNA 34a-5p, which form a toehold switch, as well as the gene of β-galactosidase. At the presence of miRNA 34a-5p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of β-galactosidase which catalyzes the substrate X-gal to produce 5-bromo-4-chloro Indigo (blue color). The mechanism is shown as Fig.2.

Fig.2 The mechanism of toehold switch-LacZ.


To express β-galactosidase in BL21 bacteria, the recombined plasmid pET-28a-toehold switch-LacZ controlled by miRNA 34a-5p was constructed using PCR method. For identification, the restriction endonuclease digestion and PCR assays were performed, which showed that the fragment length of lacZ was consistent with the expected results (Fig.3)

Fig.3 Identification of pET-28a-toehold switch-lacZ plasmid. M: Marker, 1: The plasmid of pET-28a-toehold switch-LacZ, 2: The pET-28a-toehold switch-LacZ plasmid was digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The LacZ gene amplified by PCR method.


pET-28a-toehold switch-lacZ plasmid was transfected into BL21ΔlacZ strain（LacZ deleted）. Under the optimal conditions, the cell-free expression system was prepared by mixing the cell extract with other components such as ATP, PEP, amino acid, etc. (see protocol section for details). After the filter paper was blocked with bovine serum albumin (BSA), washed and dried, a drop of the cell-free reaction system mentioned above fell onto the filter paper strip which was followed by putting it into the ultra-low temperature refrigerator and frozen dryer to form a paper strip sensor.

In order to obtain sensitive and fast detection effects, the reaction conditions that X-gal is converted to 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase in the cell-free expression system was optimized under different temperature, reaction time and miRNA concentration, which were shown as follows:

Fig.4 The optimization of reaction temperature at which X-gal is converted to 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase in the cell-free expression system. (A): OD570 value, (B): Photograph of paper strip sensor reaction in cell-free system.

Fig.5 The optimization of reaction time for β-galactosidase enzyme reaction in cell-free system. (A):OD570 value, (B): Photograph of paper strip sensor reaction in cell-free system.

Fig.6 The optimization of miRNA concentration to trigger the expression of β-galactosidase catalyzing the 5-bromo-4-chloro Indigo (blue color) production in cell-free system. (A):OD570 value, (B): Photograph of paper strip sensor reaction in cell-free system.

The optimization results showed that the best temperature is 30°C, as shown in Fig.5. When the reaction lasts for 1h, the reaction is almost over, so 1h is chosen as the best reaction time (Fig.6). For miR-34a-5p target sensor, the lowest limit of visible color development is 500fM (Fig.6).

Contribution

According to Al-Rawaf et al. [2] and Kuang et al. [3], miRNA 34a-5p was upregulated in the serum of MDD patients, as shown in Figure 1A. According to Feng et al. [4] and Kuang et al. [3], miRNA 221-3p was upregulated in the serum of MDD patients, as shown in Figure 1B. Meanwhile, Bahi et al. [5] and Mendes-Silva et al. [6] reported increased miRNA let-7d-3p concentrations in MDD patient serum as shown in Figure 1C. The above findings all support the 2022 ICJFLS team and their project.

Figure 1. (A) miR-34a-5p expression in normal and MDD cells. (B) miR-34a-5p expression in MDD cells before and after treatment

However, we also noticed that miRNA 34a-5p was downregulated in some other MDD patient tissues, including the anterior cingulate cortex (ACC) [7] and the Brodmann area [8]. This implies major differences in cellular activity and metabolism among cells of different tissues in MDD patients, leaving room for future research.

Furthermore, many other researchers have reported findings of other miRNAs in MDD patient tissues, including miR-363-5p [9], miRNA 218-5p [10], and miRNA 320a-5p [10], as shown in Figure 2. Therefore, new parts can be designed to target the above biomarkers and support MDD diagnosis.

                   
                  Figure 2. Additional mRNAs that unnormally expressed in MDD

In addition to miRNAs, several types of long non-coding RNA (lncRNA) are also expressed at abnormal levels in MDD patient tissue cells and can serve as biomarkers for the disease. Examples include TCONS_l2_00001212, NONHSAT102891, and TCONS_00019174 were downregulated, while ENST00000517573 was upregulated [11].

Additionally, many other types of molecules also exist at abnormal levels in MDD patient tissues, making them potential biomarkers for MDD. Examples include Immunoglobulin A, estrogen, serotonin, Plasma C-reactive protein, g-aminobutyric acid, and cortisol [12, 13].

This year, our YiYe-China team is utilizing the secondary structure of mRNA to diagnose gastric cancer. Through our process, we used the RNAfold website to predict mRNA secondary structures. Many research topics involve RNA secondary structures, but currently, programs such as RNAfold are not used very frequently as it is relatively new. Here, we suggest that such computer programs will provide substantial help to RNA-related research in the future.

References

1. Wan Y, Liu Y, Wang X, Wu J, Liu K, Zhou J, Liu L, Zhang C. Identification of differential microRNAs in cerebrospinal fluid and serum of patients with major depressive disorder. PLoS One, 2015 Mar 12;10(3): e0121975. doi: 10.1371/journal.pone.0121975
2. Zhou L, Zhu Y, Chen W, Tang Y. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response. J Affect Disord. 2021 May 1;286: 80-86. doi: 10.1016/j.jad.2021.02.063
3. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014 Nov 6;159(4):925-39. doi: 10.1016/j.cell.2014.10.002
4. Yakoh A, Pimpitak U, Rengpipat S, Hirankarn N, Chailapakul O, Chaiyo S. Paper-based electrochemical biosensor for diagnosing COVID-19: Detection of SARS-CoV-2 antibodies and antigen. Biosens Bioelectron. 2021;176:112912. doi:10.1016/j.bios.2020.11291