In this stage, our goal was to verify the effectiveness of a LIRA system reported in the literature and explore the best conditions and experiment setting to test LIRA before developing our own LIRA system for cancer screening ^[1]. We chose H01 and H05, two LIRAs that have been reported with good performance to conduct our verification experiments ^[1]. We first transformed plasmids that carried LIRA or target RNA of LIRA (input RNA) into BL21-DE3, and then used IPTG to induce the transcription of LIRA and input RNA, and finally examined EGFP expression in the IPTG treated BL21-DE3 with Western Blot analysis and fluorescence cytometry.

Plasmids carrying H01 and H05 LIRA were synthesized according to the sequences provided in the literature ^[1] (Figure 1).

These plasmids were transformed into DH5-α for amplification. Transformed clones were grown in LB medium. Plasmid DNA from these clones were extracted by maxiprep, and subjected to digestion with endonucleases. The digested plasmid DNA was analyzed by agarose gel electrophoresis, which demonstrated that the sizes of these plasmids were correct (Figure 2-3).

To generate clones that have both LIRA plasmids and their corresponding input plasmids, H01+input01 & H05+input05 plasmids were co-transformed into BL21-DE3, an engineered type of E.coli for protein expression. Plasmids from transformed BL21-DE3 were extracted by maxiprep, and digested by endonucleases. Agarose gel electrophoresis of the digested plasmids demonstrated that H01+input01 & H05+input05 plasmids were co-transformed successfully (Figure 4-5).

To compare EGFP expression in BL21-DE3 clones with LIRA plasmid, H01 and input01 or H05 with input05, all BL21-DE3 clones were conducted IPTG-induction of EGFP protein by culturing the transformed BL21-DE3 in LB with IPTG 0.1mM for 4h.

The transcription of H01 and H05 LIRA is regulated by T7 promotor, which requires T7 RNA polymerase. The transcription of T7 RNA polymerase in BL21-DE3 is regulated by lacUV5 promoter. The plasmid carrying H01 and H05 LIRA (pCOLADuet-1) has a gene that encodes Lacl protein, which can suppress the lacUV5 promotor. As a result, transformation of H01 and H05 plasmid into BL21-DE3 would suppress any transcription that requires T7 RNA polymerase. IPTG can combine with LacI protein, and release the suppression of LacI protein on lacUV5 promoter, which activates the transcription of T7 RNA polymerase. Therefore, we could use IPTG to induce the transcription of T7 RNA polymerase in BL21-DE3 transformed with H01 and H05 plasmid, and the induced T7 RNA polymerase could activate the transcription of LIRA and the EGFP reporter gene downstream of LIRA. After IPTG induction, the H01 and H05 transformed BL21-DE3 clones were treated by ultrasonic disruption, and cell lysate was subjected to SDS-PAGE and Western Blot analysis.

Result of SDS-PAGE on H01 and H01+Input01 transformed BL21-DE3 clones demonstrated that a strong expression of a 27kDa protein in the IPTG-induced H01+input01 clones. Result of Western Blot demonstrated that the expression of EGFP protein in H01+input01 transformed BL21-DE3 clone is much higher than that of H01 transformed BL21-DE3 clone with or without IPTG induction, indicating that Input01 RNA successfully changed the structure of H01 and exposed the RBS and start codon in H01 for translation of downstream EGFP.

Similarly, result of SDS-PAGE on H05 and H05+Input05 transformed BL21-DE3 clones demonstrated that an increased expression of a 27kDa protein in the IPTG-induced H05+input05 clones. Result of Western Blot demonstrated that the expression of EGFP protein in H05+input05 transformed BL21-DE3 clone is slightly higher than that of H05 transformed BL21-DE3 clone with or without IPTG induction, indicating that Input05 RNA might slightly change the structure of H01 and exposed the RBS and start codon in H01 for translation of downstream EGFP.

Together, while the expression of EGFP protein in H01+input01 transformed BL21-DE3 clone was much higher than that of H01 transformed BL21-DE3 clone, the expression of EGFP protein in H05+input05 transformed BL21-DE3 clone was only slightly higher than that of H05 transformed BL21-DE3 clone. Such result demonstrated that the effectiveness of H01 LIRA that targets input01 RNA is much higher than that of H05 LIRA that targets input05 RNA.

To quantify the LIRA-mediated EGFP expression, the fluorescence of protein lysate from all BL21-DE3 clones were measured by Multi-mode Microplate Reader. To compare the effectiveness of H01 and H05 quantitatively, we used the On/Off ratio. The On/Off ratio of LIRA is defined as ON-state reporter gene expression, which is LIRA-mediated downstream gene expression in the presence of the corresponding input RNA, divided by the OFF-state reporter gene expression, which is LIRA-mediated downstream gene expression in the absence of the corresponding input RNA.

The On/Off ratio of H01 is 28.554, while the On/Off ratio of H05 is 1.345. Comparing the fluorescence measurement results of H01 and H05, the On/Off ratio of H01 is much greater than H05, indicating that H01 is a much more effective LIRA. Therefore, we decided to develop our own LIRA based on the structure of H01.

Using a variety of bioinformatic analysis, our model group identified miR-142-3p and miR-210-3p as ideal biomarkers for cancer screening in post-myocardial infarction patients. We did Wound healing and Transwell assays to confirm that these two miRNAs could promote the migration and invasion of cancer cells. For detailed information of this part of work, please see our Model section.

In this stage, our goal was to verify the effectiveness of our designed double-arm LIRA that target both miR-210-3p and miR-142-3p. The double-arm LIRA has two recognition regions for two different target RNA, and an ideal LIRA could be activated only in the presence of both these two target RNA. Our model group analyzed the sequence and structure of LIRA, and used multiple indicators to calculate the expectation score of LIRA to predict its effectiveness (Please see our Model section for detailed information). We chose 3 double-arm LIRA with different expectation scores to test their effectiveness on detecting miR-210-3p and miR-142-3p.

We cloned these LIRAs into pCOLADuet-1, and named these constructs double-arm LIRA 1, double-arm LIRA 2, and double-arm LIRA 5 respectively.

All plasmids listed in Table 3 were transformed into BL21-DE3(Figure 19-21) and plasmids from successfully transformed clones were verified by digestion with restriction endonuclease and agarose gel electrophoresis (Please see our Engineering section for detailed information).

Transformed BL21-DE3 clones was treated with 0.1mM IPTG for 4h and then disrupted by ultrasonic-sound. Cell lysate was collected by centrifuging and diluted to 1.8 μg/μL. The EGFP fluorescence from the diluted cell lysate was measured at a medium-high PMT gain. The clones transformed with double-arm LIRA 1 did not have increased EGFP expression even with both input142 and input210, indicating that it could not be activated by miR-142-3p and miR-210-3p (Figure 22). The clones transformed with double-arm LIRA 2 showed increased EGFP expression even with input142 alone, indicating that it could be activated by miR-142-3p alone (Figure 23). The clones transformed with double-arm LIRA 5 showed increased EGFP expression only with both input142 and input210, indicating that it could only be activated in the presence of both miR-142-3p and miR-210-3p (Figure 24).

In the future, we would like to apply our LIRA-based screening approach to screen blood samples from post-MI patients, to identify those patients with high risk of cancer. Since it is unlikely that we could transform miRNAs from blood samples into bacteria, we need to adapt our LIRA-based screening approach into a cell-free system. In addition, for a screening approach, it would be much easier to detect changes of color than expression of EGFP, which requires machine to measure fluorescence. Therefore, we set to make two improvements of our LIRA-based screening approach. First, we change the reporter gene from EGFP to LacZ, which could hydrolyze CPRG (yellow) into CPR (purple)^[2]. Second, we use a cell-free system to express the LacZ cloned in our LIRA constructs.

To set up the cell-free system for LIRA, we first tried H01 LIRA that we have successfully validated in our experiments at the beginning. We replaced the reporter gene from EGFP to LacZ in H01 LIRA construct. For the cell-free system, we chose the PURExpress® In Vitro Protein Synthesis from New England BioLabs. With CPRG as substrate, the results of LacZ expression could be visualized. (Please see our Engineering section for detailed information)

Next, we combined our double-arm LIRA 5 with a LacZ reporter gene, and tested it with the cell-free system. We added the double-arm LIRA 5 plasmid with miRNA and CPRG into the cell-free system. After incubation at 37 ° C for 2h, the results could be visualized, and also quantified by measuring OD₅₆₂ with a Multi-mode Microplate Reader. The result demonstrated that our double-arm LIRA 5 could be activated by miR-142-3p and miR-210-3p in the cell-free system (Figure 25).