DESIGN: We can model a phosphorylation site (mostly Threonine or Serine, although Tyrosine also potentially phosphorylates) with a phosphomimetic amino acid (primarily Aspartic acid or Glutamic acid) which mimics the charge distribution or structure parameters. Thus, some Alzheimer’s marker phosphorylation can be incorporated into the protein by phosphomimetics to mimic the same conditions.
BUILD: We selected a few sites of relevance: 231T, 198S, 199S, 205S etc and replaced with Aspartic acid.
TEST: We obtained the structures using ChimeraX and AlphaFold by using energy minimisation option.
LEARN: The structures are completely different from the initial intrinsically disordered. The arising hydrophobicity can be correlated to the tendency of aggregation.
DESIGN: Proliferation of BL21 E. coli cells, and modifying the newly formed cells to make competent cells. Competent cells can take up plasmids and can express them.
BUILD: We used chemical method of producing competent cells. The preformed BL21 cells (brought from another lab) were propagated and then subjected to buffers containing RbCl, CaCl2, PIPES, etc. to make them competent to induce plasmid and express it.
TEST: Transformed plasmid, specific for expression in BL21 cells, and plated it on non-antibiotic LB agar plate. Control was setup, which involved transformation of original BL21 (from which it was propagated) with the same plasmid.
LEARN: We found that the original stock of BL21 had proper growth, which approved the functionality of the plasmid. But, unfortunately there was no growth in the prepared competent cell. This might have happened due to improper methodologies or slight difference in buffer. Utmost care needs to be taken during competent cell preparation. There is also a high chance of contamination, and it involves destruction of cells.
DESIGN: Transformation of Tau/pET29b (Addgene #16316) plasmid into BL 21 E. coli using heat shock method.
BUILD: We modified some aspects of a general heat-shock method of plasmid transformation. The protocol we followed can be found in the protocols section.
TEST: We grew the primary culture of bacteria in Kanamycin antibody. The plasmid, which has KanR gene, expresses in Kanamycin antibody.
LEARN: We saw proper growth of Bacterial culture, which meant that the bacteria successfully took up the plasmid and could express it. Afterwards we also extracted protein from it, which again proves its success.
DESIGN: After successful transformation, the bacterial culture can express the protein. The protein can be extracted and purified to obtain pure protein, free from contamination. This protein can be used further in testing and phosphorylation.
BUILD: The protein could be expressed using IPTG, in E. coli bacterial culture. We modified some aspects of a general protein purification. The protocol we followed can be found in the protocols section. We could use Ni-NTA gravity column method of purification, due to availability of His tag in our construct.
TEST: We ran SDS gel to obtain protein bands. We compared it to the standard protein marker. The protein, conjugated with His tag, needs to come at around 50kDa range.
LEARN: We could obtain a single protein band in the eluted fraction. We did find bands in the same range in Wash fraction and slightly in flow through, which concludes that this might not be the efficient method of purification, in terms of wastage. But we are sure that this is a fairly good method, in terms of the quality of protein obtained.
DESIGN: Since the aptamers bind with the protein pTau, we expect a shift or smear of the protein bands when the protein incubated with the aptamer at optimal concentration is loaded on a native gel made with TBE. Thus, if we do a WB with the aptamer incubated protein, there will be an observable compared with the case of no aptamers.
BUILD: We improvised the standard protocol of EMSA, instead of using autoradiography we used visualisation of the protein using the antibody conjugate (pTau rabbit monoclonal and anti-rabbit hrp-conjugated).
TEST: We did Western blot with the protein incubated with the aptamers at 0.1 micromolar concentration.
LEARN: We found that although the smear is evident, there is a significant background. This is because of using skimmed milk as blocking agent, which contains casein. Casein is a phosphoprotein and the pTau antibody may have some non-specific binding tendency to Casein as an Ab selected for a phosphoprotein may have non-specific binding with phosphoproteins as the phosphorylated moiety is always either of Threonine, Serine or Tyrosine.
DESIGN: SELEX relies on randomness, as the chances of an optimal sequence to bind with the target increases with large combinations. Thus, if the randomisation is done at a greater degree, in each round of SELEX on a partition of the eluded aptamers, the selection of Aptamers may be accomplished in exponentially lesser time. We found that the incorporation of non-conventional bases like dPTP and oxo-GTP can cause random point mutations at an incredible level of 10% after a few cycles of PCR followed by asymmetric PCR. We needed a high mutation rate due to small size of the aptamers.
BUILD: Since our Aptamers were already selected for the target by 17 cycles by the original authors, we planned to perform the same with a different sample DNA. However, on analysing the aptamers, we hypothesised that high G repeats may have been responsible for a better binding to the target. dPTP causes G biased transitions.
TEST: On performing the PCR, we couldn’t observe the bands of the PCR product in the gel.
LEARN: The reasons could be firstly, incorporation of non-conventional bases might slow down the action of normal Taq-polymerase. Secondly, the dPTP or oxo-GTP incorporated DNA may not be a perfect helix, as these bases exist in tautomeric form.
DESIGN: C8-Alkyne dCTP may undergo cycloaddition with VHL ligand, having an azide label. This product after HPLC purification can be incorporated into an aptamer by terminal transferase. The ligand can help localising the Aptamer around VHL E3 ubiquitin ligase, leading to polyubiquitination of the target.
BUILD: We set the protocols for accomplishing click reaction of C8-Alkyne dCTP with [S, R, S]-AHPC-PEG1-Azide (VHL Ligand). We also modelled the structure of the product.
TEST: We did the calculation of the radius of gyration and other aspects of the molecule by computer simulation.
LEARN: We got the radius of gyration to be 16.54652 Angstroms, which shows that the final molecule is highly coiled and thus, may have unexpected behavior.
DESIGN: Run MAWS on the VQIINK part of 2mz7 pdb (residues 275-280) and get an Aptamer Sequence.
BUILD: Perform the necessary improvements to the software as mentioned in the AptaLoop documentation. Create a GUI (tkinter based or web based, preferably web-based) of the software for the help of others who will use this tool. Understanding what sqm actually does, and what is exactly stored in it, along with the error messages shown by it.
TEST: Run the examples, to get an idea what is happening in each module. Chopped down 2MZ7 PDB to get the said hexapeptide (VQIINK). Added H atoms wherever was missing using PyMol to get a working.
LEARN: Residues more than 10 did not work. Infinite pause time error was occurring because of an os.waitpid() function. Duplicate atom warning was coming up. Understood which Aptamer sequence to work with given a whole pool is generated in output. Need to start building the part and integrate other tools, and also the GUI.