Part:BBa_K5492710

Y1_aptamer

Y1 ssDNA aptamer sequence designed to specifically bind to histamine. The overhang of the 5' end of the sequence is complementary to j1_y1_aptamer_connector. The connector sequence contains a 5' biotin (5'biosg) tag that lets the sequence bind to an avidin-coated magnetic 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.

Transdermal device

The transdermal device and its usage are written in our protocol in the Experiment topic under the title “Transdermal transport of the enzymes and aptamers”. Please refer that paper for details. Here we emphasize only the final measurement which is a truly simple A260 measurement. We received back from our team members three series of 300 uL volume samples which contained the aptamers packaged in liposomes and dispersed in the hydrogel. We also received two series dispersed in hydrogel without packaging them in liposomes. Each sample series consists of six 300 uL samples arriving to the acceptor phase after 1, 2, 4, 8, 12 and 24 hours.

Before the transdermal experiment we dissolved altogether 552.7 ug DNA aptamers dissolved in 1200 uL TE buffer. From this amount 200 uL was used and was evenly distributed among the five, one-day-long (24 h) transdermal experiment. This means that in each of the five experiments we used 1/5*(200/1200)*552.7 = 18.42 ug aptamers at the donor phase. We determined the DNA content of the acceptor phase by measuring A260 values with Thermofisher Nanodrop device. The results were the following:

The following graph shows the 5 data series results:

As it is clearly visible there are no precise trends in these results. Though the second experiment shows a large peak after eight hours, the other two control experiments don’t represent the same, so we can conclude that there is no evidence for a trend-like transdermal transport amongst aptamers in these experiments. It is still worth it to consider the summed-up transmission:

Considering the exact, total amount of the transmission, we can recognize that only 3-6‰ of the aptamers went through the ex vivo membrane, which means that this method is not proper topical use of the aptamers.

'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.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]