Wet Lab

Our project ExoSwitch aims to create a diagnostic tool to detect and quantify blood exosomal miRNAs using de-novo-designed riboregulators, toehold switches ¹. Previous research highlights that cancer cells express abnormal quantities of exosomal miRNAs ^2-3, we hypothesized that these exosomal miRNAs could serve as biomarkers of cancers easily accessible. To quantify these miRNAs, we have decided to adapt the toehold switches, a synthetic biology tool developed to detect long RNAs. For our proof-of-concept, we aimed to assess the specificity and sensitivity of toehold switches for oncogenic miRNA markers extracted from cancer-derived exosomes. Our strategy consisted of quantifying these miRNAs using our toehold switches and, for comparison, the gold standard method that is Polymerase Chain Reaction. We wrote a script, named SwitchMi Designer, to model a bank of toehold switch candidates targeting specific miRNAs. We finally used three different methods to produce these toehold switches.

Toehold Switch Construction

We modeled a bank of de-novo-designed riboregulators, toehold switches, targeting specific miRNAs using our SwitchMi Designer. We defined the miRNAs we wanted to detect and quantify according to the literature, and we obtained a bank of toehold switch candidates for 6 miRNAs.

Table 1: These parts are DNA fragments that encode toehold switch candidates targeting miR-141-5p. They contain the BamH1 and BglII restriction sites.
trigger miRNA	number of toehold switch candidates generated by SwitchMi Designer
miR-21-5p	7
miR-34a	3
miR-141-5p	8
miR-155-5p	3
miR-200c	7
miR-210	1

We verified the toehold switch candidates' MFE structure and free energy using RNAfold web server. Thus, we selected the candidates with the expected MFE structure and favorable free energy (higher when the toehold switch is not linked with the miRNA). We decided to focus only on the detection of 2 miRNAs, miR-21-5p and miR-141-5p that are commonly overexpressed in cancers.

We used several methods: DNA synthesis using SOE PCR, and molecular cloning using restriction enzymes and golden gate assembly, to build the toehold switch candidates targeting miR-21-5p and miR-141-3p.

DNA synthesis by SOE PCR

Since the designed Toehold Switches are roughly of 900bp (counting the repressed gene GFP), we first decided to produce them with a PCR-based synthesis of DNA sequences also called Splicing for Overlap Extension PCR (SOE PCR). SOE PCR illustrated on Figure 1 consists in the amplification and progressive elongation of the starting piece of DNA (GFP in our case) with the help of constant forward primer and variable overlapping reverse primers (oligonucleotides) (Figure 1: forward primer in yellow and backward (bw) primers in red). Adding one forward and one type of reverse primers per PCR reaction we observe splicing of the starting piece of DNA with the sequence “brought” by a reverse primer. The result of each SOE PCR reaction can be verified with an electrophoresis gel through comparing the molecular weight of the product with the weights of the control DNA pieces (starting DNA piece, and all the pieces obtained during the previous SOE PCR cycles).

Figure 1. Schematic representation of the SOE PCR technique. The set of primers (in red) consists of three overlapping sequences of nucleotides able to contribute to the synthesis of the target DNA sequence (green+blue) after three cycles of SOE PCR. Green piece of DNA taken as the starting sequence (GFP in our case) is elongated during each cycle (in the 5’-->3’ direction) after a primer’s fixation thanks to the activity of the specific PCR enzymes (TaQ polymerase in our case)

The SOE PCR method has been regularly used by the research team that provided us with a working space in the laboratory. Thus we were able to draw inspiration from their protocols as well as from the protocols described in such papers as Ai-Sheng Xiong et al. 2006⁴, TerMaat et al. 2009⁵.

We found that 3 reverse primers (of the average length 20-50bp) would be sufficient to synthesize the whole Toehold Switch sequence. The team of our supervisor Pierre Nioche provided us with the DNA sequence encoding for eGFP, thus we took eGFP as the starting DNA piece for the SOE. After designing and ordering the primers we dedicated 1.5 months of wet lab work to testing the technique. Please find the results following the link.

Protocol DNA synthesis by SOE PCR

Molecular cloning

We aimed to synthesize the toehold switch by assembling the variable part of the toehold switch (Fig. 2), into a plasmid that already encodes the repressed gene, GFP.

Figure 2: Scheme of a toehold switch. In our project ExoSwitch, we define the variable part of the toehold switch the part that differs depending on the target RNA. Indeed, the Toehold part is complementary to the target miRNA. Thus, the complementary nucleotide at the root of the hairpin differs with the target miRNA. Created with BioRender.

We used two different molecular cloning methods, one with restriction enzymes and the other one with golden gate assembly. Molecular cloning defines the insertion of a DNA fragment, called insert, into a vector such as a plasmid. In our case, the newly created plasmid is introduced into Bl21(DE3) and selected using antibiotic resistance (Fig.3).

Figure 3: Scheme of our strategy developed to build the bank of toehold switch candidates. Firstly, we engineer a plasmid encoding for GFP followed by a T7 terminator. Secondly, we assemble the variable part of the toehold switch preceded by a T7 promoter into the previous plasmid. Toehold Switch is then transcribed via T7 RNA polymerase.

We used E.coli Bl21(DE3) for their competency and the plasmid DE3 carrying the T7 RNA polymerase gene. Indeed, we engineered the toehold switches with T7 promoter and T7 terminator that are recognised by the T7 RNA polymerase for its transcription into RNA. We proceeded in two parts to build the toehold switch: the repressed gene, GFP, in one hand, and the variable part of the toehold switch in another hand. Indeed, if we have a plasmid with GFP and the T7 terminator, we can reuse it to test any toehold switch by assembling the variable part in it.

Cloning using restriction enzymes

We performed molecular cloning using type II restriction enzymes. These enzymes have the ability to cut specific DNA sites, called restriction sites. The plasmid and the insert are digested using the specific restriction enzymes before being purified on a gel. Thanks to a ligase, the insert is attached to the plasmid.

Figure 4: Creation of toehold switch targeting human specific miRvia molecular cloning using restriction enzymes. This is a two step process, starting with the assembly of GFP in pET-24d(+). It is followed by the assembly of the variable part of the toehold switch into the newly created plasmid. This plasmid encodes for the toehold switch via T7 RNA polymerase activity.

We first built a plasmid encoding for the repressed protein of the toehold switches, GFP (Fig. 4) using the EcorI and XhoI restriction enzymes.

To do so, we amplified GFP from the GFP tagged protein via PCR. We designed the primers with the EcorI and XhoI restriction sites for the PCR reaction. Then, GFP has been digested using EcorI and XhoI restriction enzymes and has been purified with gel migration. We performed the same digestion and purification step on pET-24d(+) plasmid.

We achieved the ligation step between digested GFP insert and digested pET-24d(+) using NEB T4 ligase as described in the protocol below. We then introduced the plasmid (containing the part BBa_K3878000) into E.coli 10-BETA to amplify it.

Protocol molecular cloning using Restriction Enzymes

We designed the variable part of the toehold switch candidates with the T7 promoter and the restriction sites BamHI and BglII. We ordered the variable part from the toehold switch targeting the human miR-141 on IDT. The synthesized fragments, the variable part of the toehold switch, are received into plasmids from IDT, they are resuspended in an elution buffer and amplified into E. coli 10BETA. Plasmids are then extracted from bacteria cultured in liquid TB media overnight using BioBasic EZ-10 Spin Column Plasmid DNA Miniprep Kit. We digested the variable part of the toehold switch candidates and the previously created plasmid (containing the part BBa_K3878000)) with the restriction enzymes BamH1 and BglII (Fig. 3). The digested DNAs were purified and ligated using the same protocol as before.

Golden gate assembly

Golden gate assembly is an in vitro molecular cloning method used to assemble several inserts into a plasmid. This method is based on type IIs restriction enzymes and overhangs compatibility^6-7. Type IIs restriction enzymes have the ability to cut DNA outside of their recognition sites and to generate overhangs. This method is used to assemble multiple inserts into a plasmid using sequential or simultaneous activities of a single Type IIS restriction enzyme and T4 DNA ligase. We designed the variable part of the toehold switch targeting human miR-21-5p to overlap with the overhang of the plasmid BBa_K3453101 and BsaI recognition site. Evry iGEM team kindly gave us the part BBa_K3453101 which is a plasmid engineered for Golden Gate assembly using BsaI as a restriction enzyme. This plasmid contains GFP and the T7 terminator.

Figure 5: Creation of toehold switch targeting human miR-21-5p via molecular cloning using Golden Gate assembly. The variable part of the toehold switch is assembled in a one step process via Golden Gate with BsaI restriction enzyme. This plasmid encodes for the toehold switch via T7 RNA polymerase activity.

The synthesized variable part of the toehold switch was amplified into E. coli 10 BETA. Plasmids were then extracted from bacteria cultured in liquid TB media using BioBasic EZ-10 Spin Column Plasmid DNA Miniprep Kit. The variable parts of the toehold switch were amplified via PCR technique to raise the quantity of insert as in the protocol below. Synthesized inserts are then amplified via polymerase chain reaction (PCR). We followed the protocol given by NEB® Golden Gate Assembly Kit (BsaI-HF®v2) for the assembly of one insert. (Fig. 5)

Protocol molecular cloning using Golden Gate assembly

Cell culture, exosome extraction and miRNA quantification

Cell culture

We have selected for our proof of concept two well-known cancer cell lines, MCF-7 and HeLa, respectively breast and cervical cancer cell lines, because they are reference cell lines used to study cancer⁸. Few studies described that exosomes secreted by those cell lines contains deregulated miRNAs: two miRNAs signature were upregulated (miRNA-21-5p and miRNA 141-3p) beside seven miRNAs were downregulated (let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p)^9-11 .

We used a medium based on DMEM +10% of FBS for cell culture and phosphate-buffered saline (PBS) to wash the cells and remove the dead ones. Due to the cell adhesion on the interior surface of the flask, we used trypsin to detach them and later resuspended them in the medium. This technique is used to grow the targeted cell line until we reach good confluency (between 80-90%). Cells were regularly tested negative for mycoplasma contamination, and no contamination was detected.

Protocol Adherent Cell culture

Exosome extraction and miRNAs isolation

We were able to perform exosomes extraction from the media when the cells were at 90% of confluency after 6 passages for MCF-7 cells and 3 passages for HeLa cells culture. The exosome extraction method is based on differential centrifugations with different levels of intensities going from 2000 xg to 100.000 xg rpm in order to remove each large component (“contaminations”) and isolate the exosomes. We performed BCA (BiCichondinic Assay). Western blot experiments were performed to determine whether we succeeded in our exosomal preparation test using detection of markers of exosomes, CD9 and CD63, and one negative control, calnexin.

Protocol Exosome extraction

To confirm the abnormal quantity of exosomal miRNAs secreted from cancer cells, we extracted RNA from the HeLa and MCF-7 exosomes using the Invitrogen™ Total exosome RNA and protein isolation kit. RNAs were isolated from HeLa cell exosomes by mixing the sample with a denaturation solution, Acid-Phenol, and 100% ethanol to lyse the exosomes followed by a number of centrifugation rounds and the addition of a washing solution in order to separate our RNAs from all other contents of exosomes. Finally, we had the RNAs in 100 µl of free nuclease water.

Exosomal miRNAs quantification using RT qPCR

To quantify miRNA-21-5p and miRNA-141-5p from exosome extracts, we did a quantitative reverse transcription assay by using a kit (Takarabio) Mir-X™ miRNA FirstStrand Synthesis and TB Green® qRT-PCR10. First, miRNAs were tagged with PolyA tails at their 3’ end using the previous kit to lengthen the miRNA sequences that are originally too short for qPCR. We designed specific primers to detect miR-141-5p, miR-21-5p, and miR-34a (due to their absence in HeLa exosomes). qPCR was done on one exosome extract from HeLa cells and 4 conditions are tested: miR-34a (negative control), U6 (Takara’s kit control), miR-21-5p, and miR-141-5p. In order to have a Cq (quantification cycle) measure to compare with our samples, we replace cDNA in the mix with H2O. Cq is the number of cycles required for the sample to cross the threshold line (the background fluorescence intensity). This value is inversely proportional to the quantity of RNA, which means a low value of Cq indicates a high quantity of RNA. The aim of this study is to detect miRNA in the exosome, it will be reflected by sample Cq inferior to the water Cq.

Protocol Western Blot and miRNAs extraction

Protocol miRNAs quantification using qPCR

Pardee K, Green AA, Ferrante T, et al. Paper-Based Synthetic Gene Networks. Cell. 2014;159(4):940-954. doi:10.1016/j.cell.2014.10.004
Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences. 2008;105(30):10513-10518. doi:10.1073/pnas.0804549105
Happel C, Ganguly A, Tagle DA. Extracellular RNAs as potential biomarkers for cancer. JCMT. 2020;2020. doi:10.20517/2394-4722.2020.71
Xiong A-S, Yao Q-H, Peng R-H, et al. PCR-based accurate synthesis of long DNA sequences. Nat Protoc. 2006;1(2):791-797. doi:10.1038/nprot.2006.103
TerMaat JR, Pienaar E, Whitney SE, Mamedov TG, Subramanian A. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler. Journal of Microbiological Methods. 2009;79(3):295-300. doi:10.1016/j.mimet.2009.09.015
Engler C, Kandzia R, Marillonnet S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability. El-Shemy HA, ed. PLoS ONE. 2008;3(11):e3647. doi:10.1371/journal.pone.0003647
Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. Peccoud J, ed. PLoS ONE. 2009;4(5):e5553. doi:10.1371/journal.pone.0005553
Fauzi AN, Norazmi MohdN, Yaacob NS. Tualang honey induces apoptosis and disrupts the mitochondrial membrane potential of human breast and cervical cancer cell lines. Food and Chemical Toxicology. 2011;49(4):871-878. doi:10.1016/j.fct.2010.12.010
Honegger A, Schilling D, Bastian S, et al. Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells. Wells S, ed. PLoS Pathog. 2015;11(3):e1004712. doi:10.1371/journal.ppat.1004712
Lin Y, Zhang C, Xiang P, Shen J, Sun W, Yu H. Exosomes derived from HeLa cells break down vascular integrity by triggering endoplasmic reticulum stress in endothelial cells. Journal of Extracellular Vesicles. 2020;9(1):1722385. doi:10.1080/20013078.2020.1722385
Wong GL, Abu Jalboush S, Lo H-W. Exosomal MicroRNAs and Organotropism in Breast Cancer Metastasis. Cancers. 2020;12(7):1827. doi:10.3390/cancers12071827
Schageman J, Zeringer E, Li M, et al. The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. BioMed Research International. 2013;2013:1-15. doi:10.1155/2013/253957
Cancer. Accessed October 20, 2021. https://www.who.int/news-room/fact-sheets/detail/cancer
Cancer Facts & Figures 2019 | American Cancer Society. Accessed October 20, 2021. https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2019.html
Shi M, Guo N. MicroRNA expression and its implications for the diagnosis and therapeutic strategies of breast cancer. Cancer Treatment Reviews. 2009;35(4):328-334. doi:10.1016/j.ctrv.2008.12.002
McGuire A, Brown JAL, Kerin MJ. Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring. Cancer Metastasis Rev. 2015;34(1):145-155. doi:10.1007/s10555-015-9551-7
Chen G-Q, Zhao Z-W, Zhou H-Y, Liu Y-J, Yang H-J. Systematic analysis of microRNA involved in resistance of the MCF-7 human breast cancer cell to doxorubicin. Med Oncol. 2010;27(2):406-415. doi:10.1007/s12032-009-9225-9

Team:UParis BME/Wetlab