[alexholehouse]

So it seems like there are two timescales for what 'transient' means in the paper.

1) There are different levels/numbers of i-motifs identified depending on the cell cycle position (highest at G1/S boundary, although this is only comparing cells synchronized to G1, G1/S and early S so maybe more at different points?), suggesting these structures don't just stably form and then just sit happily around for the entire lifetime of the cell.

2) In vitro these motifs are less stable than (say) G-quadruplexes so presumably there is a suggestion they may be transient over short timescales, but this is not actually examined in the paper. No idea how you'd actually test this without inherently perturbing the equilibrium being examined in vivo. Even if you could avoid fixing the cells antibodies would be out of the question because of the inherent linkage (if you bind the i-motif with a 500 pM Kd [high affinity] you're gonna HUGELY stabilize that conformation).

[subroutine]

I'm curious about this...

"What excited us most is that we could see the green spots – the i-motifs – appearing and disappearing over time, so we know that they are forming, dissolving and forming again,”

anyone know what technique they used during this observation? It sounds.. not fixed?

what matters most in my opinion is whether this is a spontaneous DNA conformation or whether this is a transient conformation DNA assumes while transcription machinery is preparing to read nearby bases. (if the former, kinda interesting; if the latter, much less interesting)

[alexholehouse]

Right - yeah; I don't know, I had the same thought when I read the commentary (I read the paper first). Nothing in the papers suggests the ability to monitor formation/loss in realtime I don't think? My guess is this is an interpretation of the data from the fact that you see lower levels in G1, higher levels in G1/S and lower levels in early S - i.e. they must be 'transient' because the levels go up and down again.

Seeing this happen in real cells in realtime would be – I would have thought – technically almost impossible. We're at the cusp of viewing the formation/loss of clusters of RNA POL or mediator clusters with the most advanced super-res (see Ibrahim Cissé's work) but these are comparatively massive protein clusters, so the idea of being able to view DNA structural transitions at [effectively] single-molecule resolution where that transition involves a few nucleotides in a non-perturbative way seems like a reach.

Seems like the obvious next step is to break 'em with synonymous mutations and ask if there's any detectable phenotype.

[subroutine]

Apparently they used ELISA.

[subroutine]

But like you said...

"No idea how you'd actually test this without inherently perturbing the equilibrium being examined in vivo. Even if you could avoid fixing the cells antibodies would be out of the question because of the inherent linkage (if you bind the i-motif with a 500 pM Kd [high affinity] you're gonna HUGELY stabilize that conformation)."

I mean, I trust the reviewers/editors of Nature but I don't understand how this is not a serious confound!

[robbiep]

The article states that they used fluorescent antibodies that bound specifically to this motif as opposed to other motifs