Tuesday, July 24, 2007

Measuring Stacking Interactions

 
The two strands of double- stranded DNA are held together by a number of weak interactions such as hydrogen bonds, stacking interactions, and hydrophobic effects [The Three- Dimensional Structure of DNA].

Of these, the stacking interactions between base pairs are the most significant. The strength of base stacking interactions depends on the bases. It is strongest for stacks of G/C base pairs and weakest for stacks of A/T base pairs and that's why it's easier to melt A/T rich DNA at high temperature. (It is often incorrectly assumed that this is due to having only two hydrogen bonds between A/T base pairs and three between G/C base pairs.)

The figure below shows a melting curve of various DNAs. The curve shows the conversion of double-stranded DNA to denatured single strands by following the change in absorbance as the temperature is increased from left to right. When the double helix is unzipped the absorbance increases. Note that poly(AT) "melts" at a lower temperature (TM = melting temperature) than poly(GC). This is because the average stacking interactions of G/C base pairs are two or three times stronger than A/T base pairs so more thermal energy is need to disrupt them.


The base stacking interactions have been measured in several different ways but most of these measurements are indirect and all of them have been with double-stranded DNA. Of the single-stranded polynucleotides, only polyA has a helical structure in solution and that's because of the stacking interactions between single adenylate resides in the polynucleotide. PolyT is somewhat unstructured and polyG and polyC have complex three-dimensional structures that are difficult to interpret.

Assuming that the stacking interactions of the adenylate residues is the only significant force maintaining the polyA helix, it's possible to measure the stacking interaction directly by pulling both ends to see how much pressure it takes to disrupt the helix. This can be done by fixing single-stranded polyA to a substrate and grabbing the other end with a molecular probe. The elasticity of the DNA can be measured by single-molecule atomic-force spectroscopy (Ke et al. 2007).

As the molecule is stretched, it resists up to the point were the bases become unstacked and the helix is disrupted. The force required can be used to directly calculate the stacking interactions between the adenylate residues. The value turns out to be 3.6 ± 0.2 kcal/mol per base (15 kJ/mol). This is very close to the stacking energies calculated for A/T base pairs in earlier experiments. (The stacking energies for G/C base pairs in DNA are about 61 kJ/mol.)

The experiment is independent, and direct, confirmation of the literature values for stacking interactions. The energies of these stacking interactions turn out to be significantly larger than the energies of the other weak interactions involved in holding double-stranded DNA together (hydrogen bonds, "normal" van der Waals interactions, and hydrophobic interactions).


Changhong Ke, Michael Humeniuk, Hanna S-Gracz, and Piotr E. Marszalek (2007) Direct Measurements of Base Stacking Interactions in DNA by Single-Molecule Atomic-Force Spectroscopy. Phys. Rev. Lett. 99:018302
[The top figure is from Ke et al., 2007]

9 comments :

  1. Huh, I never knew that was what caused the difference in melting temperature. Sure, I knew that base-stacking was important for the structure as a whole, but I didn't realize it was dependent on the sequence. Just to be clear, this means that the Tm of CGCGCGATATAT would be higher than the Tm of CACACAGTGTGT, correct?

    ReplyDelete
  2. The Tm refers to the melting temperature, which is the midpoint of the transition between the double-stranded DNA and the completely denatured molecule with free single strands.

    In your second example it's clear that the A/T rich region would separate first followed by the G/C rich region. (This is why many promoter regions tend to be A/T rich.) But it's not clear whether the actual Tm of the two molecules would be different.

    ReplyDelete
  3. I have a question: when a DNA duplex is melted or unzipped, the energy cost should include both the stacking energy between the neighboring base pairs, and the hydrogen bond energy between the disrupted bases. It seems to me your suggested experiment to measure the "stacking interactions" actually measures both the stacking energies and the Watson-Crick hydrogen bond energies. Is my understanding correct?

    Thanks!

    Jie

    ReplyDelete
  4. anonymous asks,

    I have a question: when a DNA duplex is melted or unzipped, the energy cost should include both the stacking energy between the neighboring base pairs, and the hydrogen bond energy between the disrupted bases.

    Yes, that's correct. What we'd like to know is the relative contributions of those two forces.

    It seems to me your suggested experiment to measure the "stacking interactions" actually measures both the stacking energies and the Watson-Crick hydrogen bond energies. Is my understanding correct?

    The experiment describes how to measure the stacking interactions in single-stranded DNA. This eliminates any contribution due to base pairs between the two strands in double-stranded DNA. The results suggest that most of the stability of the double helix is due to stacking interactions and not base pairs.

    ReplyDelete
  5. Many thanks! Do you know any convincing measurements of the percentage of the contribution to DNA duplex stability from stacking interaction between intact DNA base pairs?

    I ask because I am studying possible defects that can be excited in a DNA duplex. In addition to the disrupted base pairs (melted base pairs or "bubbles"), I wonder whether there are defects that can form by giving away the stacking interaction while keeping the hydrogen bonds. From the mechanical energy point of view, such defects may cost lower energy and thus more likely to form then bubbles. However, bubbles may be more twistable so bubble formation may release the twisting freedom and increase the entropy. I am studying that under certain conditions whether these too types of defects can co-exist.

    I like your blog very much!

    Best regards,

    Jie

    ReplyDelete
  6. Dr. Moran,

    Thank you so much for the nice article and your excellent explanation. Would you mind to provide a reference for "The stacking energies for G/C base pairs in DNA are about 61 kJ/mol."

    Best regs,

    ReplyDelete
  7. this paper came out a little early than Ke's PRL paper and tried to explain the percentage of contributions from base stacking and base pairing.

    Yakovchuk. Base-stacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Res (2006) vol. 34 (2) pp. 564-574

    Randy Bin Lin

    ReplyDelete
  8. Thanks for this blog. I was wondering why is the stacking interaction for poly GC stronger than that of AT. Is it just because in the GC base pair the additional H bonds decrease the flexibility and therefore entropic cost for the stacking is lowered?

    I'm new to this field of DNA. I'm a chemist by training. I'm sorry if this is a stupid question..

    Best wishes,
    N

    ReplyDelete
  9. thank you for the post. I had a professor offer this slide to our class (unmarked) and quiz us on which was which. Long after the answer was discovered, I simple did not get the concept. Thank you for the explanation.

    ReplyDelete