Stacking interactions in DNA - help needed

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MirkoZlikowski
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Stacking interactions in DNA - help needed

Post by MirkoZlikowski » Tue Mar 28, 2017 3:41 pm

Dear forum members,

I have posted this question on two other forums, but got no response. I'm starting to worry that my questions are way too dumb - if that's the case, please freely say so. If cross-posting is against the rules - my apologies to admins and please delete/ban if that happens to be the case.

Anyhow, I have a problem understanding how base-stacking interactions contribute to the stability of dsDNA. My lack of understanding is probably caused by my insufficient knowledge of basic chemistry and I was wondering if anyone here can explain, in relatively layman's terms, what base stacking is and how it contributes to the stability of double-stranded DNA. So, what bothers me is the following:
 
In various resources (books, on-line sites, etc...) which describe the types of interactions that keep the two strands of DNA together, usually three types of forces are mentioned: hydrogen bonding, base stacking and hydrophobic effects. Many people, including older ones like myself, have been taught that the two DNA strands are held together by hydrogen bonds between complementary A-T and G-C base pairs (2 bonds per A-T base pair and three per G-C base pair). However, not-so recent articles (such as "Base-stacking and base-pairing contributions into thermal stability of the DNA double helix", Nucleic Acids Res (2006) 34 (2): 564-574.), clearly demonstrate that "base-stacking is the main stabilizing factor in the DNA double helix", whereas hydrogen bonds play minor, if any role. The problem is that, after consulting several textbooks, forums, articles, etc, I still don't understand how base stacking really works.

I also have been taught that two complementary ssDNA strands, instead of remaining separated, tend to form dsDNA because in dsDNA multiple hydrogen bonds between complementary base-pairs will be formed. 
However, I later learned that when DNA is in single stranded form, bases interact with water (in aqueous solutions) and form an even greater number of hydrogen bonds with water molecules than they do when they are paired with complementary bases in dsDNA form. Therefore, formation of dsDNA from ssDNA cannot be driven by the creation of (greater number of) hydrogen bonds between base pairs, since many more hydrogen bonds between water molecules and bases in ssDNA need to be broken to form the double helix in the first place. Therefore, hydrogen bonds are not the drivers behind dsDNA formation and do not play a major role in holding the two DNA strands together - i.e. they provide base-pairing specificity for replication/transcription/recombination, but do not contribute much to the DNA stability. Is this correct?
 
Enter stacking and hydrophobic interactions.  If I understand correctly, since bases are hydrophobic, in aqueous solutions they will tend to align one over the other in order to minimize their hydrophobic surface area that is in contact with water. Bases achieve this by aligning their rings into a parallel orientation, i.e. one base more or less "sits", or stacks, on top of another (for simplicity not going into twist and roll angles, slide etc). Anyway, I think I can understand why this happens on a very primitive level - if we have two bases separated by some distance in water, there will be a cage of water molecules around them. However, if two bases stack on top of each other, water will be released from between the two stacked surfaces, which will release water molecules from an ordered system (cage) into a more disordered system (free water). So, even though we have more ordered system with respect to bases, much more water molecules become disordered, total entropy of the system is larger and base stacking is therefore entropically favored. Is this correct?
 
Given all this, my questions then are as follows:
1) Do base-stacking interactions form only in dsDNA, or also in ssDNA? Will then single-stranded DNA form stacked structures in aqueous solutions (apart from polyA, which is known to do so)? And finally, will single DNA strand by itself adopt an analogous (same?) helical structure as if it would if it were paired with complementary DNA strand? If not, then why not?

2) Second, and this one is really bugging me: How do stacking interactions stabilize dsDNA? Let's take a 2bp dsDNA molecule as an example:

5' 3'
| |
A-T
G-C                                               
| |
3' 5'
 
Here A is stacked on top of G and T is stacked on top of C. How do the 5'-AG-3' stacking and 5'-CT-3' stacking hold two single DNA strands together? I can accept that 5'-AG-3' stacking keeps A and G 'locked' on top of each other in a single strand of DNA, but how does the 5'-AG-3' stack interact with complementary (5'-CT-3') stack/strand to maintain dsDNA form, i.e. where do the stacking interactions between bases in opposite DNA strands occur to keep the two strands paired? In the above example, does A interact with C and T with G via some sort of 'cross-stacking' interactions? And are then those cross-stacking interactions what holds the two single DNA strands together? Since, as the above cited article (and textbooks, etc.) state that hydrogen bonds don't play a significant role here, as can be seen in the following sentence: "For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization". Which brings me to the following question.
 
3) Suppose we mix two non-complementary, equal length, single-stranded DNA molecules in water. From what I 'understand' - bases in those ssDNAs should also tend to "avoid" water. Will those ssDNAs then form some sort of weird structure in which bases are going to be stacked in the middle of the structure, but not form hydrogen bonds (since they're not complementary?). I am naively thinking about some sort of zipper like structure.
Or, will two single strands remain single-stranded and unstacked. If latter is true (which I don't know), then how can we claim that hydrogen bonds do not play a major role in stability and formation of dsDNA?
 
So, thanks everyone for reading this and apologies for the tremendous amounts of ignorance and confusion I demonstrated in this post. I hope someone will be able to clear things up for me. I'm offering a pizza and a beer, this has been bugging me for too long.

claudepa
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Re: Stacking interactions in DNA - help needed

Post by claudepa » Thu Mar 30, 2017 10:44 am

you are obviously already a specialist of this field. You could see also DNA intercalating agents. Some have a very high affinity for DNA.
They intercalate between base pairs and their high affinity is due to stacking forces. They are positively charged, otherwise they could not have access to DNA. I do not know if there are publications on structure (cristallography or NMR) of single strand nucleic acids but there are on the structure of abasic sites where a DNA base is missing. If I remember well generally the other base, which does not has hydrogen bonds, is still at the same location or it soutside the helix because stability is better with the stacking of the neighbouring base pairs.

MirkoZlikowski
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Re: Stacking interactions in DNA - help needed

Post by MirkoZlikowski » Thu Mar 30, 2017 12:35 pm

Hello claudepa,

Thank you for taking the time to write your answer. On a side note - I am definitely not a specialist in the DNA chemistry/(bio)physics. I just wanted to refresh my knowledge about DNA structure and I started reading several molecular biology/biochemistry textbooks, such as Biochemistry by Stryer, Molecular Biology of the Cell by Alberts et al, Molecular Biology by Cox, Dudna and O'Donnell and Understanding DNA by Calladine (and forums, and a couple of articles that are waaaaaaaay above my level, etc). As I was reading and thinking about what I've read I realized that I don't really understand, or"feel", how base-stacking operates. If I were a specialist in the field I would probably know the answer to my questions :-). That's why I was asking for a really down-to-earth, layman's type of answer. Or, to paraphrase a famous physicist, I would appreciate if someone could explain how base stacking stabilizes DNA, as simple as possible, but not simpler.

To get back to your answer, I am aware of intercalating agents, particularly of ethidium-bromide, and I understand why such chemicals show a tendency to intercalate between neighbouring base pairs in dsDNA. Unfortunately, it isn't obvious to me how intercalating agents explain the contribution of stacking to dsDNA stability. EtBr is a single entity, not attached to any particular DNA strand via N-glycosidic (or other) bond, while base-pairs at any single level along the dsDNA molecule are separate entities, each attached to its own DNA strand. And the major bond holding these two separate entities together is not hydrogen bond, but stacking. I was probably not clear enough in my original post since English is not my main language, so I hope no one will mind if I rewrite and dumb down my main question to the painful levels of ignorance:

How the hell the 'glue', called stacking, which 'glues' bases on top of each other in single strand of DNA, also glues two strands in dsDNA together. As I mentioned earlier, for the 2 bp example:

5' 3'
| |
A-T
G-C                                               
| |
3' 5'

Stacking glues A on top of G in one DNA strand, and T on top of C in another DNA strand. But how does stacking glues together two separate entities (bases comprising basepairs) in separate DNA strands: A with T and G with C. As I mentioned earlier, I thought that the glue were hydrogen bonds, but as the article I quoted states: "base-stacking is the main stabilizing factor in the DNA double helix". How? Where do the stacking interactions between two DNA strands, two separate entities, occur to allow for that fact?

claudepa
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Re: Stacking interactions in DNA - help needed

Post by claudepa » Fri Mar 31, 2017 2:56 pm

I said you are already a specialist because you took time to look your point on stacking in books and articles and you are very motivated to find the answer. Do not believe that necessarily some specialized reseracher knows the answer to your question. May be yes may be no. When I was doing my phD I found something unusual on DNA, however nobody understood and therefore gave credit to my results. Many prestigious DNA reseachers went for lectures in our laboratory and each time I was asking them what could explain my result. I had many answers but never the right one. Only I was lucky enough in the bench to find the explanation years later (After my phD, which was therefore a little bit difficult to obtain). This teached me that you have to be very patient when you are a researcher. And when you change very frequently of topics, because of funding, you might miss important results.

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