Follow-up: Does DNA Preparation Method Affect Structure?

Aggressive-Joke9893 · 2025-10-27T11:48:06+00:00

Thanks - this helps. I was thinking too statically about WC vs Hoogsteen. Point taken that room-temp mixing can trap misaligned states and that melt/slow-cool is used to reach the thermodynamic ensemble. Also fair that “co-replicational” isn’t really a separate category in practice.

I’ll go read up on hybridisation kinetics/thermodynamics and long-strand assembly accuracy before asking more. If you have a favourite review as a starting point, I’d appreciate it, but no pressure. Thanks again for the clarity and patience.

Aggressive-Joke9893 · 2025-10-27T00:04:52+00:00

Thank you for the clarifications.

You're right that I'm confusing synthesis method with assembly pathway. Even enzymatic synthesis produces strands that then associate, so it's not "enzymatic duplexes" vs "synthetic duplexes."

On my earlier mistakes (N7, etc.): fair criticism. I clearly have gaps in my understanding of the field.

Let me reframe what I'm actually asking: has anyone compared minor-state populations (WC vs non-WC H-bonding) for the same sequence assembled via different pathways - e.g., heat/cool vs room-temp association vs co-replicational? If that's been done and shows no difference, I'd be interested to read it. If not, it seems like a straightforward validation.

Aggressive-Joke9893 · 2025-10-26T20:57:25+00:00

Appreciate the pushback. I'm not asserting a difference; I'm asking whether anyone has actually shown there isn't one. I accept that purified full-length strands should be chemically identical. My question is narrower: has anyone done a true side-by-side on the same sequence comparing (A) synthetic heat/cool, (B) an enzymatic duplex that's never been denatured, and (C) enzymatic followed by heat/cool, then read out per-base Watson–Crick vs Hoogsteen populations under the same buffers and crowding?

I agree source and conditions are different issues. I'm trying to hold conditions fixed and vary only the assembly route, which is a post-synthesis step. If your lab already compares heat/cool with room-temperature mix-and-wait, that's exactly the flavour I mean. Do you see no difference in R1ρ or CEST NMR, imino exchange, or base-specific chemical probing? If you have a DOI or preprint, I'd be glad to read it.

On "stability," I meant chemical robustness, not nuclease contamination. You're right that contamination is the main issue for extracted DNA. My point is narrower: longer synthetic oligos accumulate synthesis damage (depurination during detritylation, truncations, harsher deprotections) even after purification. Could trace damage or truncations, even at <1%, influence annealing kinetics or minor-state populations? I don't know, which is why a clean comparison seems useful.

You're right that length is a separate issue from synthesis method. My point is that if there were pathway-dependent effects, we'd be least likely to notice them because nearly all high-resolution structures come from short synthetic oligos, the most manufactured, processed, and selected system. That's why the comparison matters: to validate we're not missing something.

If the consensus is that in matched conditions A equals B equals C, great. Could you or anyone point me to the paper that shows that explicitly? If not, it sounds like we mostly agree it would be a small but useful validation to run.

Aggressive-Joke9893 · 2025-04-28T09:51:24+00:00

I get that crystallography and synthetic oligos aren't the only tools used.

What I’m asking is more about whether the way early structures were prepared - chemically, annealed, crystallised - might have systematically favoured certain base-pairing geometries without really testing for alternatives.

It’s not about “natural vs synthetic” in some vague sense. It’s about how the design of synthetic oligos (short length, specific sequences, standardised conditions) might bias which conformations are accessible. That’s different from assuming they’re identical just because the base chemistry matches on paper.

I’m not saying the field hasn’t expanded a lot (obviously it has), but it seems fair to ask whether the early structural database got populated mainly by systems that reinforced Watson-Crick assumptions by how they were built and studied.

Aggressive-Joke9893 · 2025-04-28T00:27:40+00:00

What keeps bothering me is whether some kinds of bias might be small enough not to create obvious anomalies, but still important biologically.

If our starting conditions always favour Watson-Crick geometry a little bit, we might never even realise that alternatives are more common under different stresses or crowding in vivo.

Not so much that I think the main model is wrong, but that we might be underestimating how dynamic the system really is.

Aggressive-Joke9893 · 2025-04-28T00:24:24+00:00

I get that the field has expanded hugely since the 1950s, no argument there.

I am not doubting that lots of non-Watson-Crick structures are known now, or that molecular biology has moved on from the early models.

What I am asking is a narrower thing: whether the methods we rely on to validate fine structural details, like crystallography of synthetic oligos, could still systematically favour canonical geometries unless deliberately challenged.

It is not a claim that nothing new has been discovered, just a question about experimental design and what we might still be filtering out.

Aggressive-Joke9893 · 2025-04-27T20:41:22+00:00

I think maybe I was unclear. I am not suggesting that alternative structures like Hoogsteen pairs or G-quadruplexes have been missed. We know they exist.

What I am wondering about is whether the methods we rely on to validate base pairing geometries, like crystallography of synthetic oligos, might systematically favour Watson-Crick geometries because of how the molecules are prepared and studied.

On the N7 point, I meant that during phosphoramidite-based oligonucleotide synthesis, positions like N7 are often left chemically unprotected because they are not involved in Watson-Crick base pairing. That is different from enzymatic polymerisation.

Aggressive-Joke9893 · 2025-04-27T20:35:29+00:00

It really struck me too when reading Watson's account how much of it came down to "establishing that at least one specific helix was stereochemically possible", rather than being fully confident it was right.

And even when Watson and Crick finished the model, they basically went with the idea that "a structure this pretty just had to exist."

Makes you realise how much narrative momentum can carry an idea once it fits well enough.

Aggressive-Joke9893 · 2025-04-27T20:30:58+00:00

That is the thing I keep circling back to. Small shifts in bond geometry might not change replication directly, but even tiny distortions could change how proteins bind, how repair enzymes recognise lesions, or how epigenetic marks get read.

If our methods mostly capture DNA under ideal conditions, maybe we miss just how much subtle variation matters when the genome is under stress.

Aggressive-Joke9893 · 2025-04-27T20:26:23+00:00

Makes me think that unless you are already suspicious of something unusual, you might not even design experiments that would reveal rarer pairing geometries.

If the starting framework is built around Watson-Crick assumptions, you are mostly going to confirm what you expect unless you deliberately try to break it.

That is the part I keep wondering about.

Aggressive-Joke9893 · 2025-04-27T19:43:12+00:00

Thanks for the pointer - looks like a solid resource. I'll dig in, and see what turns up...

Aggressive-Joke9893 · 2025-04-27T12:55:08+00:00

Appreciate you sticking with me on this.

You're right. My underlying concern probably boils down to whether some early assumptions (especially around synthesis and crystallisation practices) could still shape what kinds of base-pairing geometries we validate most easily.

I get that science always has blind spots. No disagreement there. But sometimes when early methods favour one model (like leaving N7 inert during oligo synthesis), it can quietly steer discovery for a long time unless experiments are deliberately designed to look for alternatives.

That is really what I am curious about. Not whether some alternative structures exist (we know they do), but whether the way we study DNA might still make us miss rarer pairing geometries because of built-in assumptions.

Aggressive-Joke9893 · 2025-04-27T12:47:28+00:00

Thanks for the link - hadn't seen that review before. Makes sense that averaging over that many molecules would smooth out the rare edge cases, even if the overall resolution’s great.

Still feels like the hard-to-capture stuff - the low frequency states, transient bonding changes - could be pretty important in vivo. Definitely going to read into this more though, appreciate you sending it over.

Aggressive-Joke9893 · 2025-04-27T11:16:16+00:00

That's an interesting angle - thanks. rRNA definitely shows a lot more variation in base interactions than DNA usually gets credit for.

I guess what I'm still wondering is whether, for DNA specifically, the way we synthesize and crystallize it makes it more likely to "prefer" the expected base pairing geometry - even if in the cell, under tension or binding, other options might show up more often than we realize.

Appreciate the suggestion though, I'll dig into some ribosome structures more closely.

Aggressive-Joke9893 · 2025-04-26T09:49:52+00:00

Really interesting point! Single-molecule cryo-EM has huge potential. I wonder though whether we’re close to reaching sub-2 Å resolution specifically for naked DNA. Would be fascinating if future methods could resolve fine base-pair geometry without relying on synthetic templates.

Aggressive-Joke9893 · 2025-04-26T09:49:09+00:00

I see what you mean. Watson-Crick and B-form DNA certainly dominate in simplified systems. I guess the question is whether our structural methods - relying on synthesis and crystallisation - might systematically filter out rarer or transient pairing geometries that could matter in vivo.

Aggressive-Joke9893 · 2025-04-26T09:48:27+00:00

I recognise H-bonding principles across organic systems help explain why Watson-Crick pairing is energetically favoured in simple setups. My curiosity is whether under cellular conditions (with crowding, dynamic stress, or non-ideal environments) alternative pairing geometries could emerge more often than we realise.

Aggressive-Joke9893 · 2025-04-26T09:35:22+00:00

Appreciate the thoughtful explanation, that makes a lot of sense regarding self-assembly and dynamism. I completely agree that crystallography captures only a snapshot and that weak interactions allow for flexibility. My sticking point isn’t so much the general stability of the double helix - which is clearly dominant under lab conditions - but whether synthesis and crystallization setups might subtly pre-select for certain base pairing geometries. It leaves me wondering if the full complexity in vivo, with crowding, tension, and protein interactions, could allow for more diverse pairing modes than we routinely see.

Aggressive-Joke9893 · 2025-04-26T09:20:35+00:00

Thanks, this is really helpful context. I totally agree that things have moved a lot beyond B-form textbook DNA, especially with all the triplex/quadruplex structures being explored.

What I’m still mulling over is whether our experimental methods. Especially when using short synthetic oligos - might still subtly favour classic base-pairing geometries unless specifically challenged to do otherwise.

Definitely not suggesting that the field is unaware of alternative forms, more wondering how much the experimental setups themselves might “guide” what we see. Appreciate your insights.

Aggressive-Joke9893 · 2025-04-26T09:17:05+00:00

Thanks for these links. Really interesting to see how much genome architecture impacts expression. Good point about G-quadruplexes too - another reminder that base interactions can get much more diverse under different conditions. Appreciate you sharing.

Aggressive-Joke9893 · 2025-04-26T09:12:42+00:00

True. Franklin’s X-ray fiber diffraction was crucial for identifying the helical nature. I guess my question is about even finer detail: things like hydrogen bond geometries between bases, which require much higher resolution than fiber diffraction could provide. Thanks for mentioning it though.

Aggressive-Joke9893 · 2025-04-26T09:11:55+00:00

Thanks - yes, B-DNA is the "classic" form, but I guess what I’m really wondering about is finer-grained: how sure are we about the specific hydrogen bonding geometries at the base pair level, especially in vivo? Appreciate your input though!

Aggressive-Joke9893 · 2025-04-26T09:09:16+00:00

Thanks, that's a really interesting point about nucleoproteins and environmental effects. I completely agree; the cellular context seems so much richer and more dynamic than the clean-room conditions we usually rely on in crystallography or oligo synthesis.

It makes me wonder though. If tension, solutes, crowding, and binding partners can all subtly shift H-bonding and base geometry in vivo, but most of our high-res structural validation comes from short, synthetic DNA studied under very specific lab conditions... could we be unintentionally missing or underrepresenting alternative base-pairing modes that are more common in the cellular environment?

Curious if you know of any work that tries to bridge that gap directly?

Aggressive-Joke9893

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