Difference between revisions of "Part:BBa K5492701"

Latest revision as of 12:09, 2 October 2024

J1_Y1_aptamer_connector

J1 ssDNA sequence designed for serving as connector between avidin-coated magnetic beads and the 5' overhang of J1_aptamer and Y1_aptamer. The connector sequence contains a 5' biotin tag (5'biosg) that lets the sequence bind to the bead via avidin-biotin connection.

Usage and Biology

Aptamers are generally artificial ssDNA, RNA, or peptide oligomers which bind to specific target molecules. All ssDNA aptamers we utilise are proven to be able to bind specifically to histamine, thus preventing the binding of the molecule to a histamine receptor.

Experiments

'Fishing Method' With Aptamers

Our original idea was to create a sustainable tool for usage other than the topical methods. For example, foods / alimentary liquids may contain histamine, causing inflammatory bowel disease or symptoms. If we could prove that the chosen aptamers can bind to the histamine molecules, we would be able to avoid using DAO or HNMT enzymes, which may be harmful for many reasons. However, using aptamers to cage histamine would mean the food will contain this extra amount of DNA. Usually, it is not dangerous, but removing the aptamers from the food after they fulfil their role would be better. We found out that in these cases aptamers should be elongated with a short extra sequence. The method’s essence is that the (strept)avidin on the surface of the beads can bind biotylinated ssDNA, the sequence of which are reversely complement with the elongated part of the aptamer. When we apply strong magnetic field, we expect to gather not only the beads, but together with them the elongated aptamers, too. These aptamers can trap the histamine, bind to the magnetic beads’ connector sequence and thus the histamine itself can be purified from the liquid. This scenario is depicted on the following figure:

As our budget could not cover the enough sensitive histamine assay to test our idea, we decided to do it in two parts. Firstly, we proved that these beads can attach the elongated aptamers and secondly we proved that the elongated aptamers can bind the histamine successfully.

Bead binding experiment:

We started with fully saturating the beads’ avidin’ spaces with the biotylinated connectors in two steps. We tracked the process by A260 measurement. The experimental results are here:

Then, we used 200 uL beads (1.2 mg beads in each reaction) to 50 uL elongated aptamers. The results were the following:

This means that the average binding capacity of the ssDNA coated beads is cca. 500 ng DNA / 1mg connector-coated beads.

The second part was to prove that the elongated aptamers are capable to trap the histamine molecules. To check this hypothesis, we used three different dilutions of histamine: 1 uM; 10 uM; 100 uM. We added 50 uL of these solutions to 200 uL elongated aptamer solutions of which the concentration was 600 ng/uL. During the experiment we followed the procedure described in the Yokobayashi paper with two important modifications: After checking the folding of our aptamers via Mfold we recognised that the aptamers are more stable in solutions with high Mg2+ concentration. Therefore we used Thermofisher Dreamtaq Buffer with high MgCl2 concentration instead of the buffer proposed by the paper.

We used 65°C as unfolding temperature instead of the paper’s 55°C. The reason for this is that we learned from Mfold that the elongated aptamers have higher Tm in themselves. Finally, the cooled down solutions were used in the MAK432 colorimetric Histamine Assay Kit (from Sigma Aldrich). The allocation of the plate wells was the following (A1-A2 to H1-H2 were the standards; H10; H100 and H1000 represents reference with 10; 1000 and 1000 uM histamine solutions. Y=Yokobayashi a9 aptamer; J=Johnson’s H1 aptamer; S= Sullivan’s H5 aptamer – all in elongated form (see the registry for sequence details.)

And the numerical results:

Based on the above results it is obvious that in the 100 uM histamine solution the aptamer could trap the cca. 80% histamine in all three types of the aptamers. This means, that the protocol is useful and based on the used concentration we can estimate that 120 ug can trap 5 nmol histamine successfully. Thus, we could prove in two steps that our “fishing” procedure is a viable idea to use in the future to remove histamine from alimentary fluids.