Difference between revisions of "Part:BBa K5492701"

Revision as of 11:23, 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.