3 ms·

Seems like these "i-motifs" form transiently during transcription. If they are making the argument these motifs exist much longer than the course of transcript

Seems like these "i-motifs" form transiently during transcription. If they are making the argument these motifs exist much longer than the course of transcription I'd like to how DNA could remain stable in such a conformation. There are molecular machines inside nuclei that really dont like single stranded DNA, and will chomp it up on sight. If the complementary strand to the i-motif isn't itself moonlighting as some double stranded motif, how is it protected from these dnases. If they are only saying the conformation exists while DNA is undergoing transcription... this isnt exactly on par with a A-,B-,Z-DNA conformations. The primary source is behind a paywal for me (while off campus), but I'll be interested to read more about this later: https://www.nature.com/articles/s41557-018-0046-3 https://www.nature.com/articles/s41557-018-0046-3 (also, how do you confirm the existence of transient structures using fluoro antibodies? 'fret'? 'fish'?)

alexholehouse 9y ago
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 9y ago
  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 9y ago
    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 8y ago
      Apparently they used ELISA.
      - subroutine 8y ago
        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 8y ago
    The article states that they used fluorescent antibodies that bound specifically to this motif as opposed to other motifs

alexholehouse 9y ago

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 9y ago
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 9y ago
  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 8y ago
    Apparently they used ELISA.
    - subroutine 8y ago
      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 8y ago
  The article states that they used fluorescent antibodies that bound specifically to this motif as opposed to other motifs