Hello! I am new to AFM and am trying to analyze a DNA origami structure created by my colleagues. I am currently using a FlexAFM in air, dynamic mode, with a Tap150Al-G cantilever to visualize our DNA origami sample, but I'm facing some challenges. The sample is a hexagon made of six equilateral triangles, each with a side length of 40 nm, resulting in an 80 nm diagonal. The protocol I'm following for fixing the sample on mica is as follows: I apply 10 µL of the sample (origami in buffer) to freshly cleaved mica, let it sit for 7 minutes, then wash it with ultrapure water to remove excess salts, and finally let it dry in the air for 10-20 minutes.
However, the images I obtained (attached) are not what I expected. Could I have damaged the origami during the sample preparation process? I’m unsure if it’s relevant to mention, but we used only half of the scaffold to create the hexagon, with the other half potentially appearing in the background.
José Elias Abrão Neto Thank you for your response. I managed to understand why the DNA appears unassembled. It seems that exposing the DNA origami to water for an extended period causes the structure to denature. I plan to try visualizing it in a buffer solution and hopefully, I will obtain better results.
Yes, DNA origami is supposed to be principally not stable in air.
Surely, this must depend a lot on the surface, on which it is fixed, and presumably on the counterions (it might help to pretreat the mica with an Mg salt).
Mica, btw, has flat terraces of several microns or more, and "flat" is indeed atomically flat (in air and in water).
I have observed aggregating and degrading behaviour in DNA Origami when improper drying is carried out, its also mentioned in the following paper: doi:10.3791/52972 (see figure 4). You mention air drying for an extended period of time post washing, you could replace this with a quicker drying step using a gentle stream of inert gas to quickly remove the excess water and reduce the chances of aggregation and degradation.
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