Team:CSMU Taiwan/Results

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Definition for Abbreviations

Abbreviation of protein:

NPA: Influenza A virus (A/Michigan/297/2017(H1N1)) nucleocapsid protein (NP) gene, complete cds
NPB: Nucleoprotein for Influenza B virus (B/Colorado/16/2017) nucleoprotein (NP) gene, complete cds
HA1: hemagglutinin of Influenza A H1N1 (A/New York/18/2009)
HA3: hemagglutinin of influenza A H3N2 (A/Perth/16/2009)

Abbreviation of aptamers:

Apt: aptamer
Apt- NPA: aptamer which binds to NPA
Apt- NPB: aptamer which can bind to NP
Apt-HA1: aptamer which can bind to HA1
Apt-HA3: aptamer which can bind to HA3

Abbreviation of the SELEX experiment result:

Ft: fluid collected in the tube after the incubation of SELEX
W“i” (or w“i”): fluid collected after mixing with washing solution. “i” indicates the number of washing round
E“i” (or e“i”): fluid collected after mixing with elution buffer. “i” indicates the number of eluting round
NPA-“i”: the serial number “i” of the Apt-NPA of all the clones we obtained
NPB-“i”: the serial number “i” of the Apt-NPB of all the clones we obtained
HA1-“i”: the serial number “i” of the Apt-HA1 of all the clones we obtained
HA3-“i”: the serial number “i” of the Apt-HA3 of all the clones we obtained

Expression Result

As mentioned in the Experiment section, the production of protein can be roughly divided into three parts: The expression of E.coli, the purification of expressed proteins, and the dialysis and concentration of purified protein.

Part 1. Expression

After expressing our target protein( Protocol), the products were centrifuged to separate the solubles and precipitates. The samples were then checked by SDS page.(Figure 1)

Most of the production are found in S and S1, indicating that the expressed proteins are highly soluble. The dark band also indicates that both of them are the main product of E.coli expression.

Estimated Protein Molecular weight:

NPA:Sequence length 1494bp→498 codon +6 Histidine
110Da average/per amino acid→55440Da=55.4kDa
NPB: Sequence length 1680bp→560 codon +6 Histidine
110Da average/per amino acid→ 62260Da=62.3kDa

1-a :NPA expression, the framed band is our target NPA protein, approximately 55kDa.
1-b :NPB expression, the framed band is our target NPA protein, approximately 65kDa.

Most of the production for both the NPA and NPB are in the soluble solution, indicating that the proteins are soluble.

As seen in Figure 1, many other non-target protein exists in the solution. Hence, purification is taken out in the next step.

Part 2. Purification

We purified our target protein using S1 from the previous step. ( Protocol)The samples were then checked by SDS page and Western Blot. From SDS page, we are able to confirm if we removed other proteins and purified our protein thoroughly.(Figure 2)

From the SDS page, we can see that most of the target protein are found in Elution buffer, whereas the other proteins has been washed down during the purification steps, left in L, FT, and W.

2-a: An obvious band appears from E1~E8 as framed at 55kDa, the estimated molecular weight of NPA. Barely any other band appear in the elution solutions, indicating that most non-target proteins has been washed down.
2-b: An obvious band appears from E1~E5 as framed at 65kDa, the estimated molecular weight of NPB. Barely any other band appear in the elution solutions, indicating that most non-target proteins has been washed down.

Therefore, we confirmed that we purified our target protein thoroughly. Still, we performed Western-Blot to double check whether the product on the estimated sites were our target protein or not.

We added six His-tag to our target protein. Hence, from western blot, we are able to confirm if the proteins we purified are our target proteins by using antibodies who recognize His-tag. The primary antibodies bind to protein with His-tags, secondary antibodies then binds to primary antibodies, and can be recognized by Fluorescent dye. (Figure 3)

From Western-Blot, we can see that the bands were the same sites shown in the SDS-page.(Figure 2) This proves that the purified protein in the elution sites contains His-tag.

3-a: An obvious band appears from E1~E8 as framed at 55kDa. Indicating that the proteins on the correct site are our target NPA protein.
3-b: An obvious band appears from E1~E5 as framed at 65kDa. Indicating that the proteins on the correct site are our target NPB protein.

We have confirmed that the main products in the eluting solution are our target protein.

Part 3. Dialysis &Concentration

The high concentration of imidazole contained in the protein after purification could cause protein self-degeneration or aggregation, effecting the incubation between protein and Carboxyl gel in the next step. Moreover, the concentration was too low to be efficiently used. Thus, we dialyzed and concentrated the purified protein via Dialysis tube.

Comparing Fig.4-a and Fig.4-b, the concentration has become higher for more efficient use.

This proved that our method was successful.

Part 4. Optimized sequence

Since the protein yield was low and wasn’t able to supply the need for SELEX , we applied some online tools to optimize the sequence.For example, ATGme to replace the rare codon into the ones that E.coli use , in addition, to examine unexpected restriction sites. After this program, the protein yield became larger as seen in Figure 5 and Figure 6.

Comparing Fig.6-a with Fig.6-b, it is obvious that the yield of protein increased tremendously.

Aptamer Selection Result

Part 1. Evolution of SELEX protocol

Phase 1: SELEX tube

As indicated in the reference papers, a large amount(0.5~1 mg ) of target is required for this method. After three months of protein production, we finally had 0.9 mg of NPA protein to carry out this experiment.

Unfortunately, we did not dialyze our produced protein at that time, 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 a next round SELEX gel preparation. Therefore, we made two alternations to solve this issue:

Improve our protein yield by adjusting the sequence(see Modeling)
Seek for other ways to take out SELEX.

However, we still collected useful data regarding to the best environment for aptamer refolding in this experiment.

The amount of the by-products :

When aptamers refold in the ice , there were less by-products than when they refold in room temperature.(Figure 7) Thus, we kept the aptamers on ice after degeneration in the following experiments

Phase 2: 96 well plate

In the purpose of reducing the usage of protein for SELEX, we looked over papers and found some papers that perform SELEX in 96- well plate required much less protein.

However, the result for this method did not work well in our experiment(Figure 8). We tried two methods as described in the Experiment section, but nothing appeared in the gel electrophoresis after PCR.

In conclusion, we assumed that insufficient mixing between the aptamer pool and coated protein limited to 96-well plate could lead to separation of all aptamers before Washing.

Phase 3: SELEX in Eppendorf

We concluded that:

1. A smaller operation space for SELEX can reduce the usage of protein.
2. Sufficient mixure between aptamer pool and target protein is required
3. It is more predictable and easy to control/track when protein are incubated with the Carboxyl Gel.

Thus we used Eppendorf as our SELEX tube, and successfully got the following results mentioned in Part 2.

Part 2. SELEX result

A. SELEX

The protocol of SELEX(protocol) involves three solutions: flowthrough, washing, and elution, which respectively include aptamers that didn’t bind to anything, aptamers that bound to the Gel but not the target protein, and aptamers that bound to the target protein.
Unspecific Test:
To ensure that all aptamers we acquired from the elution bound to target protein instead of the Carboxyl Gel, we performed unspecific binding test. The protocol was identical to the normal SELEX, just that no protein were incubated on the Gel.(Figure 9)

We concluded that aptamers that bound to Carboxyl Gel were all washed down during the washing steps.

In our experiment, Two peeks appear after each round of SELEX, respectively after the washing and eluting step, which indicates the complete separation between the aptamers that didn’t bind to the protein incubated on the gel(washing) or the tightly bound aptamers (elution). (Figure 10).

After practicing several rounds(5~6) of SELEX, we chose the eluting peeks as our selected aptamer pools, which were expected to have high specificity towards our target protein, and were then sent to be sequenced.

The following are the aptamer pools we chose to clone in the next step

Fig 11. Gel electrophoresis result after SELEX round 6 of NPA

Fig 12. Gel electrophoresis result after SELEX round 6 of NPB

Fig 13. Gel electrophoresis result after SELEX round 6 of HA1

Fig 14. Gel electrophoresis result after SELEX round 6 of HA3

Part 3. Qualitative analysis

A. TA cloning

Aptamer pools were cloned into pGEM-T vectors with TA cloning kit, and were amplified and ligated with pGEM-T vector transformed into E.coli DH5α cells. Clones obtained were screened with M13 forward and reverse primers.
The ones that were at the length of 268np (181+87) indicates that the sequence has been successfully transformed into E.coli (Figure 15), and these positive colonies were then sent to be sequenced.

B. Sequence analyzation

Sequence were analyzed via NCBI Basic Local Alignment Search Tool, BLAST.
- NPA PDF
- NPB PDF
- HA1 PDF
- HA3 PDF

C. ELISA-Characterization of selected aptamers

The titer value of selected aptamers were determined by ELISA.
Pre-progress:
Amplify the sequenced DNA by PCR, using primers labelled with Biotin to product aptamers with biotin marker.
Two types of ELISA were practiced: Non-competitive ELISA and Competitive ELISA.
Purpose:
To confirm the titer value of aptamer towards target protein. According to the coated protein, we are able to test two important character we intend to understand about the selected aptamers:

The Affinity→ by coating target protein
The Specificity→by coating un-target protein.

Non-competitive ELISA:

Method:
Create a series of protein with different concentration via serial dilution. Coat these protein respectively to 96-well microplate. After blocking, add primary antibodies, the selected aptamers in this experiment. The following steps are the same as described in the Protocol.
Expectation:
If the affinity was high enough, as the concentration of target protein rises, the bound aptamer would increase too, resulting in the darker color presented.
Result:
We found out that the result for NPA, NPB and HA3 were more significant. Following are their data:

Apt-NPA-5 had higher affinity toward NPA protein and lower affinity toward NPB, which indicates its high specificity . As a result ,we chose them as our sample in the next test scrip part.

Competitive ELISA:

Method:
Coat the protein to 96-well microplate, each well containing the same amount of proteins. After blocking, add primary antibodies, the selected aptamers, at the same time, add protein in different concentration as competition. The following steps are the same as described in the Protocol.
Expectation:
If the affinity was high enough, as the concentration of competition protein rises, the amount of bound aptamers would decrease, resulting in the lighter color presented.
Result
We found out that NPA had the most significant result. The followings are its data: NPA(Figure 21)

Conclusion:We concluded that NPA-4, NPA-6 had outstanding affinity and specificity towards its target protein. Therefore, we chose them as the sample in the next part.

Part 4. Future work

Other aptamers failed to perform good affinity or specificity. This could be explained by the biotin labeled mechanism: Since only one of the strands of amplified aptamers was labelled with biotin during PCR, only 50% of biotin was able to bind to the right strand that had the function to distinguish the target protein. To solve this problem, we are going to use forward primer labeled with biotin to acquire data in the future.

Test Strip Result

As mentioned in the Experiment ,we tried two different methods:

Direct-Competition:
1. Method:
  0.25ul of Apt-NPA-4and Apt-NPA-5were coated on the NC paper as test lines. Then 120ul of samples containing 400X diluted 0.84mg/ml NPA protein were loaded onto sample pad.
2. Expectation:
  One site of NPA in the loaded samples should bind to Apt-NPA-4-Nanogold when flowing through release pad, while another site of NPA should bind to Apt-NPA-5 coated on the NC paper. Therefore, the test line should present the strawberry color of Nanogold particle.
3. Result:
  As seen in Figure 22, no color was shown on the test line.
4. Future prospects:
  There are two possible reasons why no color was presented: one is that only one of the two strains aptamers bound to NPA, another is that concentration on the NC paper was too low. We intend to try these conditions in the near future.
Sandwich Competition:
1. Method:
  0.25ul of NPA(1.94mg/ml)was coated on the NC paper as test line. Then 120ul of samples containing 400X diluted 0.84mg/ml NPA protein were loaded onto sample pad.
2. Expectation:
  NPA in the sample should bind to Apt-NPA-4-Nanogold when flowing through release pad,. Therefore, the higher the concentration of NPA was in the sample, the lighter the color of test line would be.
3. Result:
  As seen in Figure 22 ,a light circle of strawberry red was shown on the test line.
  We then tried other conditions including:
  - Different concentration of NPA on the NC paper
    Purpose& Method: Because the test line color was too light in the previous experiment, we tried to increase the concentration of NPA on NC paper by dropping more NPA. In other words, we added more drops, while every drop remains 0.25ul .
    
    Result:
    
    The color of test line became darker as we add 1 drop, 2 drops and 3 drops. The color remains almost the same after adding 3 drops.(Figure 23)
    Conclusion: Adding 3 drops of 0.25ul NPA(1.94mg/ml) was the most suitable condition in our experiment.
  - Different concentrations of NPA in the sample
    Purpose& Method:
    We applieddifferent dilution fold to NPA in our sample to look for the threshold concentration, the concentration that competes all the aptamers away, resulting in the fade color of test line.
    
    Result:
    The color turned lighter when the concentration of NPA rises. The darkest was PBS, then 800 fold diluted, and the lightest was 100 fold diluted. However, color still exists under this condition.(Figure 24)
    
    Conclusion: This double-proved our experiment was successful. Color goes lighter when the concentration of target protein rises
  - Different concentration of Apt-NPA-4 conjugated with Nanogold particles.
Full Wavelength Scan
Purpose& Method:
To confirm that Apt-NPA-4 has successfully conjugated to Nanogold particle, we tested Apt-NPA-4-Nanogold under Full Wavelength Scan. Blank using Nanogold particles to remove background of Nanogold absorbance.

Expectation:
Aptamers are ssDNAs, therefore if aptamers were conjugated to Nanogold, a peak would be anticipated to show up at 260nm.

Conclusion:
1ug, 2.5ug and 5ugof Apt-NPA-4 conjugated to Nanogold all showed a peak at 260nm. Indicating that three of them are all successfully conjugated.