Team:CSMU Taiwan/Experiments

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Part I. Protein production

Aptamer, the sequence of DNA, RNA, or peptide that could form a specific functional structure and bind with other molecular, protein, even whole cell, were chosen to be the sensor for distinguishing the type A and B influenza from each other. The production of aptamer need series of selection and examination. In this page, all methods that we ever used in experiment are presented.

During the aptamer selection, large amount of nucleocapsid protein of type A influenza (or NPA) and nucleoprotein of type B influenza (or NPB) were needed as target to select aptamers that can distinguish them from each other (hereafter refer to AptNPA and AptNPB). For the stable supply of NPA and NPB used in SELEX, the target sequences with plasmid were inserted into E.coli expression system to produce protein products continuously.

At beginning, target sequence was sent into pET29a plasmid and controlled by lac operon. These plasmids were sent into competent cell BC21(DE3) by heat shock process. After successfully transformed the E.coli, these bacteria would be selected by adding antibiotic kanamycin in culture process. Only those bacteria that had pET29a plasmid with target gene would be provided the antibiotic resistance and survived in selection.

The survived E.coli would become miniature factory to produce the target protein. The IPTG was added to turn on Lac operon in E.coli expression system, thus the recombinant protein would be synthesized and purified by following steps:

1. Induce E.coli to produce recombinant protein by adding IPTG in culture process.
2. Break the cell with ultrasonic processor and collect the cell lysate.
3. Centrifuge to separate and collect the suspension from lysate, because our target protein is water soluble.
4. Purify the target protein from lysate.
5. Dialyze the protein to remove imidazole.
6. Finally, get the protein product for SELEX.

To improve the yield of recombinant protein, the optimization of protein production was executed in different periods of experiment. The improvement of method in every period is descripted as below.

Phase 0: Polyhistidine-tag on target protein

Before starting producing target protein, finding an approach to identifying and separating NPA and NPB from various bacterial proteins is necessary for target protein production. Polyhistidine-tag (or His-tag) is an amino acid motif added at the terminus of target sequence. Expressed His-tagged protein can be detected or separated by the histidine residues on it. This is a cheap and convenience way for researcher to purify the recombinant protein.

The His-tag was added at the C-terminus of the sequence, and the His-tagged protein was purified by Ni-NTA column (see Fig. 1). With this method, lots of money could be saved in purification and detection process.

Figure 1.The Ni-NTA column that used in recombinant protein purification.

Phase 1: IPTG condition test

The IPTG effect in the protein expression was tested during protein production period. The E.coli was divided into groups according the time and concentration of IPTG treatment. The bacteria culture curve of all group was measured by spectrophotometer, and protein productivity was determined by western blotting.

Phase 2: Sequence optimization

The productivity of NPA and NPB was always low even after adjustment of induction condition. Therefore, the sequence of NPA and NPB was suspected to be the reason of low expression of recombinant protein. Website tools such as ATGme and SignalP was applied to optimize sequence of target protein. It can be divided into three section:

1. Removal of rare codons.
2. Detection of restriction site.
3. Prediction the existing probability of signal peptide.

The introduction of these tools work and the reports of optimization are presented in Model page. After sequence optimization, the new sequences were synthesized and used to restart the protein production process. The productivity of recombinant protein that produced from new sequence was compared with the productivity of product from old sequence. As showed in Results, the protein productivity is significantly improved in optimized sequence.

Phase 3: New method of dialysis

At early period of experiment, the dialysis membrane was used to remove imidazole remained in purification process. This method took very long time, and the protein was unstable in high concentration of imidazole for long period of time. As a result, our research team usually wasted much time at dialysis step and got poor product recovery rate.
To solve this problem, new method by ion exchanging column was applied to replace the original method by dialysis membrane (see Fig. 2 ). Unlike the original dialysis method using concentration gradient to exclude the imidazole out, the new method sorts out the imidazole by centrifuge and preserves the protein on membrane. Thus, it takes less time than before and concentrate the protein product at the same time. This method improves the experiment efficiency dramatically.

Figure 2.The Ni-NTA column that used in recombinant protein purification.

Part II. SELEX

The aptamers that can detected biomarkers of influenza was selected from random oligonucleotide pool through SELEX. The pool of ssDNA sequences that containing 40 random sites in 87 nucleic acids long was used as probe to react with the NPA, NPB, and the hemagglutinins from H1 and H3 subtype of influenza (hereinafter referred to as HA1 and HA3).

The SELEX brief steps are described below:

1. Binding: The ssDNA library is renatured to form tertiary structure in appropriate condition, and react with target protein in binding buffer.
2. Washing: Exclude unbound ssDNA with washing buffer.
3. Elution: Elute and collect the ssDNA binding on target with elution buffer.
4. Amplification: Denature the elution product and amplify by PCR process. The PCR products become library in next round of SELEX.

(This SELEX process will repeat 6-8 time to get effective aptamer. )

Just like process protein production, there had arisen bottlenecks with experiment of SELEX. Several examination and adjustments of SELEX method were executed to solve problems in different periods.

Figure 3. The schematic diagram of SELEX process.

Phase 1. SELEX in column

In original protocol, the ssDNA probes react with target protein in the special column. As indicated in the reference research, a large amount (0.5~1 mg) of target protein is required for this method. After three months of protein production, 0.9mg of NPA protein was produced to carry out this experiment.

Unfortunately, our produced protein did not be analyzed at that time, thus resulting in the rapid separation between our gel and protein due to the high concentration of sodium solution. It was going to take another long period of time before having enough protein for the next round SELEX gel preparation. Therefore, two alternations were executed to solve this issue: one was to improve our protein yield by optimizing the sequence, another was to seek for other ways to carry out SELEX.

Phase 2. SELEX in microplate

For the purpose of reducing the usage of protein for SELEX, another approach to execute SELEX in scant target protein was necessary. We found a novel method referred by article on Nucleic Acid Therapeutics. According the research, the aptamer selection was executed in microplate, and requires much less protein than using SELEX tube.

This method was carried out by using NPA as target protein, and the steps is described below:
(All buffer used in steps are the same as described in the Protocol of SELEX. )

Phase 2-1

1. Target Protein solution preparation: Take 7.5ul of NPA protein(1.22mg/ml), add into 450ml binding buffer(PBS 0.01M).
2. Coating: 2 well of the 96-well microplate was coated with 200ul target protein solution per well (4 ug protein/ per well) at 4℃ overnight.
3. Blocking: Add 200 ul of 3% BSA to the 2 wells with NPA and other 2 blank wells at 37°C for 2 hr.
4. Incubation: The library was denatured at 95℃ for 15 min, cooled immediately in ice for 10 min, and transferred to BSA-blocked blank wells maintained at 37℃ for 40 min. The uncombined ssDNAs were subsequently transferred to the wells coated with NPA at 37℃ for 40 min. (Fig. 4)
5. Washing: Add 200ul of Washing buffer with Tween 20 for four times.
6. Elution: Add Elution buffer and incubate for 90 sec, then transferred to a clean Eppendorf.

(The following steps including prescription, PCR and gel electrophoresis are the same as mentioned in SELEX.)

However, nothing appeared in the gel electrophoresis after PCR, we thus suspected that aptamers could have all bound to BSA during the first incubation step. Therefore, several alternations were carried out and be tested as bellow:

Phase 2-2

1. Target Protein solution preparation:Take 13.5ul of NPA protein (1.22mg/ml), add into 850ml binding buffer(PBS 0.01M).
2. Coating: 4 well of the 96-well microplate was coated with 200ul target protein solution (3.8 ug protein/ per well) at 4℃ overnight.
3. Blocking: Add 200 ul of 3% BSA to the 4 wells with NPA and other 2 blank wells at 37°C for 2 hr.
4. Incubation The library was denatured at 95℃ for 15 min, cooled immediately in ice for 10 min, then transferred to the 2 BSA-blocked blank wells and other 2 blank wells maintained at 37℃ for 40 min. The uncombined ssDNAs were subsequently transferred to the wells coated with NPA at 37℃ for 40 min.( Fig.4 )
5. Washing: Add 200ul of Washing buffer with Tween 20 for twice.
6. Elution: Add Elution buffer and incubate for 90 sec, then transferred to a clean 1.5 ml tube.

(The following steps that including prescription, PCR and gel electrophoresis are the same as mentioned in SELEX.)

Figure 4.The schematic diagram that showed the approach to select aptamer in the microplate.

Unfortunately, still nothing appeared in the gel electrophoresis after PCR. There are two possible reasons, one is that aptamers bound to the plates or BSA, which wasn’t quite possible. Another is that aptamers didn’t have sufficient reaction with the coated protein limited to the reaction surface of 96 well microplate.

Phase 3. SELEX in 1.5ml centrifuge tube

To solve the problem of reaction efficiency, 1.5 ml centrifuge tube was chosen to be the container of SELEX reagent and 3D rotary mixer was used to improve the mixing efficiency.
Finally, the result of DNA concentration in elution product meeting our expectation after series of adjustment of method. Our experiment could be carried on and would not be limited by the protein yield. (the method is presented in Protocol

But the good circumstances didn’t last long, the by-product started appearing in PCR result after 2-3 rounds of SELEX and impacted normal 87nts product yield seriously.

Phase 4. PCR condition optimization

Because of the by-product effect, all attempting to get a normal aptamer was failed. To find out the reason, all variables that can be control in PCR process were tested. Eventually, we summarize all data and build the PCR model of SELEX. Our finding is showed in the PCR condition optimization.

Part III. Analysis of aptamers

After aptamer selection and sequencing, it was necessary to analyze the binding ability and specificity of aptamer-target binding. ELISA (enzyme-linked immunosorbent assay) was used to test the affinity of our aptamers. The following description is about the method to analyze the properties of aptamer:

Phase 1. Non-specificity ELISA

Standard ELISA method can be distinguished into two types: specificity ELISA (also called “sandwich” ELISA) and non-specificity ELISA. Because we didn’t have the antibody of any target protein, we chose non-specificity ELISA method to analyze the production of SELEX. ELISA was executed in the steps listed below:

1. Coat the target protein in sequence dilution to the wells of microplate.
2. Aptamers with biotin are added into wells to react with coated protein.
3. Streptavidin with HRP is added to binding the biotin.
4. TMB is added to react with HRP and display blue color.
5. After fixed period of time, add HCl to end the reaction and turn the color of blue into yellow.

( HRP: horseradish peroxidase; TMB: Tetramethyl Benzidine Dihydrochloride )

The different proteins were coated in wells for different test. For example, we could get the affinity of AptNPA with its target by testing the binding ability with NPA, and know the specificity of them by testing the binding ability with NPB.

Phase 2. Competitive ELISA

After getting result from specificity ELISA, we used competitive ELISA method to get more precise result about the sensitivity of our aptamer. The protein was coated in wells in constant concentration. And added free protein in different concentration as competitive protein. This way could solve the problem that protein can’t fully coat on the surface, and get more accurate date.

Phase 3. Test Strip Experiment

After testing the titer of our aptamers via ELISA ( see Result ), the characters of our selected aptamers have been confirmed. The Ap-NPA-4, Ap-NPA-5, Ap-HA3-4, Ap-HA3-6 had the best affinity and specificity among them. Next step, we chose Ap-NPA-4 in the first test scrip tryout.

The Ap-NPA-4 was conjugated to nanogold ( as presented in Protocol ), then two methods were tried out as described below. The detailed device design and concept can be seen at Design.

Figure 5.The schematic diagram that presented how the test strip works

1. Direct-Competition:
   
   

   1. Add 8ul of ApNPA-4-nanogold to release pad, and dry it at 37℃
   2. Add 0.25ul of ApNPA-4/ApNPA-5 on NC paper as test line, and dry it at RT.
   3. Resemble the whole device as shown in Fig.5.
   4. Drop 120ul of sample onto sample pad and see its result

   
   


2. Sandwich Competition:
   
   

   1. Add 8ul of ApNPA-4-nanogold to release pad, and dry it at 37℃
   2. Add 0.25ul of NPA(1mg/ml) on NC paper as test line, and dry it at RT.
   3. Resemble the whole device as shown in Fig.5.
   4. Drop 120ul of sample onto sample pad and see its result

   
   

The result of test strip is presented in Results.

References

1. Hamula, C. L., Peng, H., Wang, Z., Tyrrell, G. J., Li, X. F., & Le, X. C. (2016). An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes. Methods, 97, 51-57.
2. Aswani Kumar, Y. V. V., Renuka, R. M., Achuth, J., Venkataramana, M., Ushakiranmayi, M., & Sudhakar, P. (2018). Development of hybrid IgG-aptamer sandwich immunoassay platform for aflatoxin B1 detection and its evaluation onto various field samples. Frontiers in pharmacology, 9, 271.
3. Kukol, A., & Hughes, D. J. (2014). Large-scale analysis of influenza A virus nucleoprotein sequence conservation reveals potential drug-target sites. Virology, 454, 40-47.
4. Li, W., Feng, X., Yan, X., Liu, K., & Deng, L. (2016). A DNA Aptamer Against Influenza A Virus: An Effective Inhibitor to the Hemagglutinin–Glycan Interactions. Nucleic acid therapeutics, 26(3), 166-172.
5. Bitaraf, F. S., Rasooli, I., & Gargari, S. M. (2016). DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX. European Journal of Clinical Microbiology & Infectious Diseases, 35(3), 503-510.
6. Tolle, F., Wilke, J., Wengel, J., & Mayer, G. (2014). By-product formation in repetitive PCR amplification of DNA libraries during SELEX. PloS one, 9(12), e114693.
7. Xu, H., Mao, X., Zeng, Q., Wang, S., Kawde, A. N., & Liu, G. (2008). Aptamer-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis. Analytical Chemistry, 81(2), 669-675.
8. Cruz-Aguado, J. A., & Penner, G. (2008). Determination of ochratoxin A with a DNA aptamer. Journal of agricultural and food chemistry, 56(22), 10456-10461.
9. Duan, Y., Gao, Z., Wang, L., Wang, H., Zhang, H., & Li, H. (2016). Selection and identification of chloramphenicol-specific DNA aptamers by Mag-SELEX. Applied biochemistry and biotechnology, 180(8), 1644-1656.
10. González, V. M., Martín, M. E., Fernández, G., & García-Sacristán, A. (2016). Use of aptamers as diagnostics tools and antiviral agents for human viruses. Pharmaceuticals, 9(4), 78.