Several students have written to me with questions about the structure of DNA. The most troubling questions are from students who have read the article I wrote about a paper that measures the stacking interactions in polynucleotides [Measuring Stacking Interactions]. In that posting I wrote ...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].THEME
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.)
Deoxyribonucleic Acid (DNA)Many students have written to say that my statements contradict their Professors and their textbooks. I'm not surprised. The old-fashioned view of DNA denaturation (pre-1990) supposed that the differences between A/T rich DNA and G/C rich DNA were due to the extra hydrogen bond in G/C base pairs. Many of the Professors who teach introductory biochemistry aren't aware of the fact that this view is incorrect. Even more surprising, some of the current textbooks have not bothered to update their material on the structure of DNA.
Here's the story as we know it today. For those students who have written to me, I repeat the caution I mentioned in my reply to you—be sure to check with your Professor before you write any tests. Make sure he/she understands why you are contradicting what was said in class so you don't get marks taken off. It's always better to do this in advance instead of arguing your case after you have lost marks on the test.
Let's look first at what happens when DNA is denatured by raising the temperature.
As the temperature increases, you start to get local unwinding of the double-stranded DNA. This unwinding occurs preferentially in regions where the two strand are held together less strongly. In these regions the strands separate to form bubbles of single-stranded regions. The DNA sequence in these regions is enriched in A/T base pairs because the interactions between the two strands are weaker in A/T rich regions. In G/C rich regions strands are held together more strongly so they don't unwind until higher temperatures. Incidentally, even at normal cell temperatures the DNA "breathes" and local regions become temporarily unwound. As you might expect, A/T rich regions are more likely to open up than G/C rich regions. This is one of the reasons why transcription initiation bubbles and DNA replication origins are often A/T rich. It's easier for the proteins (RNA polymerase, and origin binding proteins) to create the locally unwound regions.
When all of the base interactions are broken, the two strands separate. This is called denaturation. (Local unwinding is not denaturation.)
The base are now exposed to the aqueous environment. Single-stranded DNA is more stable than double-stranded DNA at higher temperature. Note that the edges of the bases will still form hydrogen bonds in this situation. They form hydrogen bonds with water molecules. In fact, they will form many more hydrogen bonds with water than they would form with complementary bases in double-stranded DNA.
As the temperature is lowered, the double-stranded form becomes more stable than the single strand in solution, and the DNA renatures. The first step is a nucleation event where two complementary regions come into contact. Nucleation is the rate-limiting step in renaturation. Once nucleation occurs, the rest of the molecule zips up pretty quickly.
It's easy to follow the denaturation of DNA because there's a difference in the absorbance of ultraviolet light between single- and double-stranded DNA. Single-stranded DNA absorbs more strongly.In a typical melting curve, you measure the increase in UV absorbance as the temperature increases. This tracks the unwinding and denaturation of DNA. The melting point (Tm) is the temperature at which half the DNA is unwound.
DNA that consists entirely of AT base pairs melts at about 70° and DNA that has only G/C base pairs melts at over 100°. You can calculate the Tm of any DNA molecule if you know the base composition. The simplest formulas just take the overall composition into account and they are not very accurate. More accurate formula will use the stacking interactions of each base pair to predict the melting temperature [Wikipedia: DNA melting].
The question is why is there a relationship between the base composition of DNA and the stability of the double-stranded regions?
The first people to think about this question didn't really understand the role of stacking interactions between base pairs in the middle of double-stranded DNA. They also didn't really appreciate hydrogen bonds. They naively assumed that the differences between G/C rich DNA and A/T rich DNA was due to the fact that G/C base pairs have three hydrogen bonds and A/T base pairs have only two [The Chemical Structure of Double-stranded DNA].
We now know that this explanation doesn't make sense. There is no net loss of hydrogen bonds when DNA is denatured, quite the reverse in fact. There are more hydrogen bonds formed between the bases in single-stranded DNA and water molecules than between base pairs in DNA. There's no reason why single-stranded DNA would renature if formation of double-stranded DNA was driven by the creation of hydrogen bonds between base pairs. For every hydrogen bond between bases you would have to break almost two hydrogen bonds to water molecules.
The most important interactions in double-stranded DNA are the stacking interactions between adjacent base pairs. You can think of this as the interactions of electrons on the upper and lower surfaces of the rings that form the bases. There are ten possible interactions between adjacent base pairs. The energies of these interactions are shown in the table on the left. The arrows indicate the direction of the DNA stand from 3′→5′ [The Chemical Structure of Double-Stranded DNA].
Note first of all that the strength of these stacking interactions (about 30 kJ mol-1 on average) are greater than the
Secondly, note that stacking interactions involving G/C base pairs are stronger (more negative) than those involving A/T base pairs. This is why the melting temperature of DNA depends on the base composition. It's not because G/C base pairs have one more hydrogen bond than A/T base pairs, it's because G/C base pairs form stronger stacking interactions.
This is why you can calculate a more accurate melting temperature for oligonucleotides if you use the stacking interactions. It's stacking interactions that determine the stability of double-stranded DNA and it's stacking interactions that are disrupted as the temperature increases and more thermal energy is added to the molecule.
Finally, the paper that I discussed in July [Measuring Stacking Interactions] measured the stacking interactions in single-stranded DNA (poly A). As it turns out, the stacking interactions between single bases are, in some cases, strong enough to force single-stranded DNA into a helical structure. This is further evidence of the importance of stacking interactions in conferring stability to the double helix.
1. The stability conferred by each hydrogen bond is the difference between the strength of the bond in double-stranded DNA and its strength in when bonded to water. Hydrogen bonds between bases and water molecules typically have strengths of about 25 kJ mol-1 and hydrogen bonds between base pairs are a bit higher.
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