RNA Aptamer Probe Influenza Detector

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igem18.cu@gmail.com

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CUHK, Sha Tin, NT, Hong Kong

CUHK Igem 2018

Results

1. in vitro Probe Screening

Aptamer Folding Assay

After generating 4 aptamers with BLOCK-iT RNAi Designer for H1, H3, H7, N1, N2, N9, Polymerase Basic 2 (PB2) each, we performed aptamer folding assay by mixing 1uM probe and 1uM 22-bp target together in aptamer folding buffer with fluorogen DFHBI. The fluorescence intensity were detected by CLARIOstar microplate reader at 447/501 Ex/Em. At least 1 aptamer from each subtype yield a statistically significant on/off ratio from using student's t test, except for H1.

fig1.1 — Fig 1.1. Aptamer refolding assay for aptamers of H1, H3, H7. A: aptamer; A+T: aptamer-target RNA pair; +ve: positive control with miniSpinach. 0 aptamer-target pairs for H1, 1 for H3 (purple) and 2 for H7 (blue and orange) had significantly positive on/off ratio. N=3. *:p<0.05, **:p<0.01, ***:p<0.005, ****:p<0.0001.

fig1.2 — Fig 1.2. Aptamer refolding assay for aptamers of N1, N2, N9. A: aptamer; A+T: aptamer-target RNA pair; +ve: positive control with Spinach. 2 aptamers for N1 (blue and purple), 2 for N9 (blue and orange), and 1 for N2 (orange) had significantly positive on/off ratio. N=3. *:p<0.05, **:p<0.01, ***:p<0.005, ****:p<0.0001.

Fig 1.3. Aptamer refolding assay for aptamers of PB2. A: aptamer; A+T: aptamer-target RNA pair; +ve: positive control with Spinach. 2 aptamers for PB2 (blue and green) had significant positive on/off ratio. N=3. *:p<0.05, **:p<0.01, ***:p<0.005, ****:p<0.0001.

2. Characterization of Aptamer

1. Specificity

To test if our probes are specific to their targets, we hybridized the probes with both their supposed targets and other targets. Microplate reader data shows that the aptamer-target pairs are significantly orthogonal.

Fig.2.1.1 Heatmap showing the microplate reader data from hybridizing different targets and aptamers. This shows that our aptamer-target interaction pairs are orthogonal. N=3.

Fig.2.1.2. Two-Way ANOVA of the data. It shows that signals are changed the most by Aptamer-Target Pairs.

2. Detection Limit

For our probe to detect minimal amount of viral RNA, it is necessary to investigate their limit of detection, as defined by the minimum amount of target RNA required to generate signal that is larger than its aptamer only (-ve) signal + 3 standard deviations. It can also be subjectively determined by visibility, though it might be less valid. Minimum amount of target RNA required to obtain a visually distinguishable difference between positive and negative signal was investigated by using 2 model aptamer probes, N2-694 and N9-545, as they represent a less sensitive and a noisier probe respectively. Reaction mixture was set with 2uM probe but in different concentration of target (i.e. 1.5uM, 1uM, 0.5uM, 0.2uM, 0.05uM).

Visually, for N2 probe, more than 0.2uM of target is necessary to visualize the difference, while ~0.2-0.5uM of target is required for N9 probe visualization. (See Fig 2.2.1 and 2.2.2)

Fig 2.2.1. Detection limit of N2 probe. 2 uM probe were added to each tube except blank. Blank = buffer + DFHBI.

(Up) Bar chart of fluorescence signal by N9 probe-target pairs, using CLARIOstar plate reader with Ex/Em 447/501. Red line indicate the smallest target concentration that form a fluorescence signal significantly larger than the signal arising from aptamer alone by plate reader.

(Down) Picture taken by ChemiDoc Imager under SYBR Green mode with Blue Trans Light Excitation.

Fig 2.2.2. Detection limit of N9 probe. 2 uM probe were added to each tube except blank. Blank consisted of only buffer, DFHBI and nuclease free water.

(Up) Bar chart of fluorescence signal by N9 probe-target pairs, using CLARIOstar plate reader with Ex/Em 447/501. Red line indicate the smallest target concentration that form a signal significantly larger than the signal arising from aptamer alone by plate reader.

(Down) Picture taken by ChemiDoc Imager under SYBR Green mode with Blue Trans Light Excitation.

3. Ions Dependency

Different concentration of sodium, potassium, calcium and magnesium ions were added to the aptamer folding reaction mixture of N2-694 and N9-545 probes, mimicking the addition of nasal fluid to our freeze-dried aptamer kit by household users. These 2 aptamer probes behaved similarly in different selected ions concentration. In the range of the ionic composition of nasal fluid, they performed well for sodium, potassium ions and magnesium ion, though an increase in calcium ions concentration will result in a decrease of fluorescence signal of both probes.

Overall, the aptamer system would not affected by sodium, potassium and magnesium ions in nasal fluid, except calcium ion. Note that the aptamer folding buffer used composed of 10mM Tris-HCl, 100 mM KCl, 5 mM MgCl2.

fig2.3.1 — Fig.2.3.1. Sodium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes. Dark blue arrow indicate the approximate sodium ion concentration presented in reaction mixture after the addition of nasal fluid.

fig2.3.2 — *Fig.2.3.2. Potassium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes.*

fig2.3.3 — *Fig.2.3.3. Calcium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes.*

fig2.3.4 — *Fig.2.3.4. Magnesium ion dependency of N2-694 (left) and N9-545 (right) aptamer probes.*

3. Optimization of Aptamer Assay Condition

We were able to monitor the change in fluorescence signal and the time required for signal development using real-time PCR system. N9-694 and N2-545 probes were used.

It was shown that our aptamer-probe pairs generally require only 10 minutes of cooling to give out a detectable signal, which is around 75 degrees Celsius. (Fig 3.1)

Fig.3.1 Time and Temperature for signal development of N9 and N2 probes. Fluorescence intensity is measured in Bio-Rad Real-Time PCR system. Reaction mixtures are heated at 95 degrees Celsius for 5 minutes, then slowly dropped to 25 degrees Celsius. N=1.

On the other hand, after refolding is completed, we performed melt curve analysis with the same real-time PCR system. Surprisingly, the melting curve shows that the fluorescence intensity drops quickly at 18-35 degrees Celsius, which does not correspond to the previous data. However, this might be caused by the assymetry of association and dissociation kinetics of DFHBI docking, which was never studied in the previous literature. Another interesting point is that both miniSpinach and aptamer-target pairs share 2 melting temperatures, suggesting a two-step mechanism of DFHBI docking. Nevertheless, the temperature where the probe achieves optimal performance is around 18 degrees Celsius, while room temperature incubation can also achieve a very goof fluorescence signal, which is user-friendly (See Fig 3.2)

Fig.3.2 Melting curves of N9 and N2 probes. Fluorescence intensity is measured in Bio-Rad Real-Time PCR system. After refolding, the reaction mixtures are heated from 4 degrees Celsius to 95 degrees Celsius over the course of one hour. N=1.

4. E. coli Expression Test

When we co-expressed the probes and targets in E. coli BL21 Star (DE3) provided by [https://2018.igem.org/Team:Hong_Kong-CUHK/Collaborations NUS Singapore-A team], we did not observe the expected ON/OFF ratio as seen in the in vitro experiments. While we initially hypothesized that this might be due to the low expression level of the probes and the target, as the positive control had low fluorescence comparing to blank as well, we performed total RNA extraction and used the RNA amount that is equivalent to the cell number for in-cell assay. To our surprise, we observed a 7-fold recovery of the fluorescence level of the positive control in total RNA, while none of that was observed in the probe + target pairs. Based on the results, we concluded that while aptamer folding without tRNA scaffold is unfavorable in E. coli, probe target hybridization is inhibited by the terminator following the aptamer. Please note that the design of this assay is following Ong et. al. 2017, showing that the data from the paper might not be accurate. However, due to the lack of time, we did not perform further alterations to improve the design (Fig. 4).

Fig.4 E. coli Probe expression test. (a) Results from initial probe screening using E. coli BL21 (DE3), data retrieved from microplate reader. N=3. (b) Relative fluorescence of total RNA and its comparison to other screening approaches. N=1.

5. Improve a previous biobrick and project – RNA Reporter for Promoter Activity Measurement

We have cloned the lpp promoter-conjugated new RNA reporters into pSB1C3 for assay, including tRNALys3-miniSpinach, tRNALys3-iSpinach-D5 and tRNALys3-dBroccoli. The overnight culture was directly used for DFHBI incubation and plate reader measurement. We found out that all 3 probes are brighter than the original biobrick (Fig.6.1).

Fig.5.1 Spinach Aptamer Reporter Test in DH5a. N=1.

Collaborating with [https://2018.igem.org/Team:Hong_Kong-CUHK/Collaborations NUS Singapore-A team], they have cloned the same constructs and performed the assay with BL21 Star (DE3), a RNase-E deficient strain. They also performed the assay at different incubation temperatures to see the temperature sensitivity of the RNA reporter. For convenience, only tiSpinach was tested. It is found that tiSpinach is less temperature-sensitive than tSpinach2.1 (Fig.6.2).

Fig.5.2 Spinach Aptamer Temperature Sensitivity Test in BL21 Star (DE3). (a) iSpinach is brighter than Spinach2.1 across all temperatures, although only at 45 degrees Celsius is the difference significant. (b) Signal-to-noise ratio calculated from dividing positive signal to untransformed signal. (c) Multiple t test showing that iSpinach is less significantly different for 37 vs 45 comparison than Spinach2.1, showing that it has higher reliaility in reporting heat-induced promoter activity. N=3.

In conclusion, we have constructed new RNA reporters with better properties than BBa_K1330000.

Team:Hong Kong-CUHK/Results