Folding Suppression Experiments

A quick recap: of the pores using the old NPN batch, 2 of them showed folding suppression. The first one I was able to collect events for 1000 and 2000 bp dsDNA, bnoth of which showed almost no folding, but had an unusually low event rate. The second suppressed folding for the first half of the experiment, clogged for 10 minutes, and allowed folding after the clog cleared. Once again, the event rate was abnormally low compared to other similar pores which did not suppress folding. Both of these experiments are mentioned in the recently submitted manuscript.

I started using the new small-pore NPN (with some of the old 1-micron hole chips that I still have a few of). The first experiment was unremarkable, and showed folding as per usual. I have yet to verify that the folding fractions are consistent with expectations, but at first glance it appears to be.

The second experiment exhibited folding suppression, but once again, only during the first half of the experiment. Below I show max blockage as a function of experiment time, showing near-complete folding suppression for the first ~1500 seconds, with folding appearing thereafter.

Once again, the event rate for this pore is extremely low (and I had to run at 400mV as a result), but notably, the event rate approximately doubles during the second part where folding is present. This time around, there was no obvious event to precipitate this change. The event rates are 0.02 and 0.04 Hz/nM for the folding-suppressed and folding-allowed parts respectively, compared to about 0.1Hz/nM for the first experiment which showed no folding suppression. After seeing this 3 times, I’m comfortable saying that this is a pattern and not coincidence.

Going back to the previous experiment which showed this 2-part behavior, I note that the event rate was also higher in the folding-allowed section, but in that case only by 30%.

This is only 1 experiment with the new devices, but getting folding suppression on my second try is encouraging that it is more likely with this new NPN than with the old batch. I’ll keep at it. In the meantime, here’s what we know about fold suppression:

1. It is a binary effect. Either it happens or it doesn’t. Only one pore exhibits lower levels of folding than the average, but even that is barely significant.
2. It relates to a static property of the device, not the DNA being used. We got folding suppression on 1000 and 2000 bp dsDNA on the same pore.
3. It relates to a currently uncontrolled property of the device (the local NPN distribution in the vicinity of the sensing pore being the most likely).
4. All pores that show folding suppression also show very low event rates (by a factor of ~10). This suggests that the hypothesis in our paper is on the right track – small active NPN pores or very few active NPN pores can account for this.
5. In both devices where folding started out suppressed and was then allowed, the event rate increased during the folding-allowed part of the experiment. I don’t yet have a good explanation for this.

I still don’t have a definitive answer, but the clues are piling up.