Team:NUDT CHINA/Proof
PAGE STILL UNDER CONSTRUCTION
Proof of Concept
To validate and demonstrate our design, miR let-7a, as an important serum biomarker for non-small cell lung cancer, was chosen as the target miRNA. Previously, miR let-7a has been reported to be down-regulated for 20%-40% in serum samples from NSCLC patients compared to healthy people 1.
AS A PROOF OF CONCEPT, let-7a was diluted in DEPC-treated water on various concentrations to assess the reliability, sensibility and specificity of our scheme.
To begin with, four different probes were designed to be probe candidates for the RCA reaction based on let7a sequence and probe design principles. Once they have been synthesized, sealed and purified (Figure 1A and B), RCA reactions with 10nM let-7a input were performed against all four probes to select the optimal probe for further test (Figure 1C and D). Electrophoresis results showed that prob1, prob3 and prob4 were all functional for the RCA reaction once sealed, among which, prob1 showed the strongest ability for such reaction. Prob1 was then selected as the probe in the further experiment.
(Figure 1)
By using this probe, the sensibility and specificity of RCA reaction were then determined with sybr I fluorescence assay (Figure 2). MiR let-7a, as the target miRNA was DISOLVED IN DEPC-TREATED WATER for various concentrations to determine the sensitivity of RCA reaction (Figure 2A). The best reaction time under such circumstance was determined to be 120min through a Real-time fluorescent assay using Sybr I as the Fluorescent dye indicating the dsDNA amount in reaction solution (Figure 2B and C). The sensitivity of such system was estimated to be under 1 fM building on a plotting the ΔFI data against minus logarithm of let-7a concentration and its fitting curve (Figure 2D). A brilliant specificity of RCA reaction was also shown by evaluating the RCA reaction strength under the input miRNA of let-7a, let-7c, let-7f and let-7g (Figure 2E).
(Figure 2)
Moreover, N-sHdC and C-sHdC protein were expressed and purified from E.coli. The expression and purification was verified through SDS-PAGE and Western blots (probed with an anti-His-tag antibody) (Figure 3).
(Figure3)
Once purified, fusion proteins were subsequently used for the dCas9 binding process together with an in vitro expressed sgRNA. TMB substrate was used to test the HRP activity (Figure 4A). Through which, the protein concentration was optimized (Figure 4B) and the basic concept of our design was proofed (Figure 4C).
(Figure4)
The detailed results could be found on the RESULT PAGE.
Reference
1 Jeong, H. C. et al. Aberrant expression of let-7a miRNA in the blood of non-small cell lung cancer patients. Mol Med Rep 4, 383-387, doi:10.3892/mmr.2011.430 (2011).
