Difference between revisions of "Part:BBa K5492722"

Latest revision as of 13:09, 2 October 2024

S1_aptamer_with_fluorophore

s1 ssDNA aptamer sequence designed to specifically bind to histamine. The sequence contains a fluorophore molecule at its 5' end (5' Cy5™). s1_reverse_compliment_strand is complementary to this sequence, which contains a quencher molecule (3'Iowa Black® RQ) on its 3' end, which absorbs the light emitted by fluorophore until the aptamer binds histamine.

Proposed Folding:

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

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.

Fluorescent assay with aptamers

The Yokobayashi paper (reference of y1_aptamer) founded a new idea to measure target concentration with its fluorescently marked aptamer. The main concept is that if we add a fluorophore (e.g. Cy5) on the 5’ end of the aptamer and inhibit its fluorescence with a quencher linked to the 3’-end of a short complementer DNA sequence, we can expect a competition between the complementer DNA strand and the histamine’s binding to the aptamer. This because if the short sequence is attached to the aptamer it is not able to fold to trap the histamine. In these cases the fluorescence will be low, while in the presence of the histamine the quencher-carrier short strand detaches from the aptamer, as the dG is more negative for the histamine-aptamer complex than the aptamer quencher strand complex. You can see this story on the following figure:

To ensure optimal environment, the Yokobayashi paper suggests a special buffer. As we were not able to get HEPES buffer, we substituted it with the similarly composed Thermofisher Scientific DreamTaq PCR-buffer. We also ensured enough high quencher DNA-strand concentration to make the reaction competitive. Following the recipe of the Yokobayashi paper we used 50 uL, 5.5 nM fluorophore-marked aptamers and 50 uL, 550 nM quencher DNA strands. Finally, we added 10 uL of the histamine solutions with different concentrations. In contrast with the paper, we used 65 C instead of 55 C, as defolding temperature. The fluorescence was triggered by 648 nm excitation and detected at 668 nm. We used three parallels and calculated the average results, which is depicted in the following table:

It is evident that the best performer in our test was the Jonhson’s H1 aptamer, which followed a constantly increasing fluorescence with the increase of the histamine concentration as you can see on the following graph.

If we assume that a measurement failure could have happened at the histamine concentration of 6 uM, we can conclude that the Yokobayashi aptamer follows the competition rule, too. Our experiments proved that the fluorescence method to assess aptamer efficiency can be very useful in many cases. The drawback of the method is the expensive fluorimeter which is not present in each laboratory. All in all, we can conclude that though aptamers cannot go through the skin they are still useful options to decrease certain fluids’ histamine concentration. We could prove this statement in our second and third experiment clearly. Refer to our team wiki.