Measurement and Theory of Hydrogen Bonding Contribution to Isosteric DNA Base Pairs
By Jean Carlos Rivera
There is conflicting evidence on the extent of the interactions of a Thymine (T) like compound, 2,4-difluorotoluene (F), when it is substituted in place of T in the sequence of a DNA duplex. In F the carbonyl groups that function as hydrogen bond acceptors in T are replaced by fluorine, resulting in a less polarized structure with extremely similar size and shape. Some crystal structures of F substituted DNA duplexes reveal that the distance between Adenosine (A) and F is relatively short, implying the possibility of hydrogen bond formation between F and A, while others show a larger distance implying little to no interaction. It has also been shown that the substitution of F for T was unfavorable to the helix, a result that was contrasted by experiments that showed that DNA polymerase handled F efficiently. The larger chloro version of F, 2,4-dichlorotoluene (L), was also handled extremely well by the polymerase, a result that was puzzling as well. So, in order to completely understand the effects of these substitutions, the groups of Wheeler, Houk and Kool undertook the impressive set of experiments and simulations described in this article.
To understand how base pairing (and mismatches) affect the stability of the DNA duplex, they performed extensive UV-Vis melting experiments. They measured the melting point of a wide variety of sequences, 12 and 14 nucleotides long, introducing one and two mismatches respectively. The data obtained from these experiments allowed them to determine the thermodynamic parameters of the melting/hybridization of each sequence, allowing them to assess the overall contribution of each mismatch to the stability of the DNA duplex. They obtained similar results using Isothermal Titration Calorimetry on selected sequences.
The stabilization that each base pair confers to a DNA sequence can be dissected into π-π stacking interactions with adjacent base pairs and hydrogen bond formation between complementary nucleobases. The extent of these interactions can be appropriately measured by terminal base pairing (TBP) measurements.
Their results show that F destabilizes the DNA duplex, and looses any selectivity towards A. This is because hydrogen bond formation between F and A is weaker than the interactions of A with the solvent. Formation of the duplex implies placing F in close proximity of A stripping it of solvent interactions, an action that results in an overall destabilization of the duplex. This destabilizing interaction between F and A is also highlighted by the TBP experiments. Finally, they used computational methods to determine the energetics of each base pair they formed during the melting experiments. The measured pairing energy was stripped down to its components using smaller molecules with similar motifs, allowing them to determine the major energetic contributions of the recognition motifs in each base pair.
In the last part of the article, they proceeded to explain why there are different crystal structures for similar base pairs. One possibility they suggest is that the F-A base pair can effectively “breathe” which causes the formation of crystals with different distances between the nucleobases. They also explain that the apparent recognition of F and L by the DNA polymerase is due to the protein’s ability to recognize the shape of the nucleobase instead of hydrogen bond complementarity.
With regards to the narrative, I find that the article was very well written. The only flaw I can point out is that it becomes a bit repetitive towards the end. This is mainly because they state their findings in the introduction, the results section and in the discussion. The figures were nice, the graphs really helped understand the data presented in the tables. In my opinion a graphical representation of the TBP results would have been better than the table they presented. Overall I think this is a really nice article, although a bit too fundamental for my taste.
Supramolecular Space 
In this study Khakshoor, Wheeler, Houk and Kool tested the capability of 2-4-difluorotoluene to form a hydrogen-bonded complex with adenine in DNA. This was done because X-ray crystallographic studies of F in DNA/RNA have shown the possibility of hydrogen bonding with the natural bases. The study of the pairing stability and selectivity of F and another similiar compound, L, in DNA was studied by a computational analysis as well as experimental. The van’t Hoff approach and isothermal titration calorimetry were used to do thermodynamic studies of duplex formation. UV-Vis melting experiments helped in the determination of the thermodynamics of the system. In the experimental procedure it was shown that F-A pairing in the DNA is destabilizing by 3.8 kcal/mol when it is compared with T-A pairing. Other findings were that pi-pi interactions contribute to the stabilization. The interactions of the system were studied by terminal base pairing measurements. F destabilizes the DNA duplex, as a consequence it has less selectivity towards A. It was found that at the end of a duplex F-A interactions are repulsive while T-A interactions are attractive. F was selective towards adenine. Computational results complement the experimental results. It was found that the interaction of F-A arises from the CH—N interaction instead of the expected F—HN interaction. L-A pairing was found to be weaker than F-A.
Overall, the article was well written and the figures complement the data and narrative for a better understanding. The introduction does a great job to begin the “story” and the conclusion wraps it up. Jean’s synopsis is great, he summarized all the studies and explained a little why those are the results. The picture I don’t know, it promotes violence?
Kool, Wheeler and Houk give us experimental and computational studies regarding the interaction and stability of 2,4-difluorotoluene (F) with adenine in the DNA, replacing thymine. The main focus of this studies is to measure and determine whether F can form a strong and stable hydrogen-bond with adenine, considering the fact that previous studies, some classify the bonding ability as zero and others as moderate, even in X-ray crystallographic studies.
For their studies they decide to study and compare the pairing stability and selectivity of F and L (dichlorotoluene deoxyroboside) with adenine in DNA. The results of the studies showed that F -A pairing in DNA is destabilizing by 3.8 kcal/mol in comparison with T-A pairing. Also, F-A interaction is repulsive, rather than attractive. When comparing results, L showed to be less destabilizing and rather more selective towards adenine. Computational studies gave rise to the fact that the interaction seen between F and adenine are seen more due to the interaction between CH-N , rather than F-HN.
I think this paper was interesting and showed how not all hydrogen bonds are the same in stability and interaction, that the elements involved play a key role in their formation. I liked the fact that they were able to join and complement experimental studies with computational studies to obtain more data and therefore elaborate a more profound and complete discussion of results.
Jean’s synopsis was really helpful and captured the essential components of the article, as well as his opinion. The picture is very creative and fun, as usual.
A very important collaboration is presented in this article when thermodynamic experiments are used with computational studies to answer a question from x-ray crystals presenting difluorotoluene in DNA/RNA indicating possible hydrogen bonding interactions between F and A in duplexes in a DNA polymerase active site. They use halogenated isosteres F, L and 23L to study the energetic contribution of different base pairs. Not only hydrogen-bonding interactions were measured, but also the selectivity of the base pairing in terms of energetic cost was studied. In brief, according with the experimental and computational data, base pair interaction T-A is more stable than F-A. However, the chlorinated L was less destabilizing than the F isostere.
More over, we found here how the “Watson & Crick” H-bonding interactions are not the main driving force for these base-pairing interactions to occur. The rigidity imparted by the geometry of this interaction id the key to understand this effect. For that reason, you can see how 23L is very unstable according with the experimental and computational thermodynamic data. They also mention the solvation effect in Adenine can explains these energetic differences. I think also, that the time of the duplex formation can provide some information of this effect. There’s an hydrophobic component of the base that prefer to buried inside the hydrophobic core of the DNA. But, those halogenated parts of the isostere can interact with the water, and according with that energetic preference of interaction with water, the information in terms of energetic stability can be obtained. In addition, some computational data of the hydration energy of these base pairs will be useful to explain this behavior in solution. Maybe minor modifications in these base-pairs can’t show the energetic effect of the whole DNA, but multiple mutations of the same base pair-isostere can offer information of the role of the waters solvation of not only Adenine but for the isostere in study.
About the effect of L against F in terms of energetic stability, I think that more than the electronegativity, the orientation and electron movement of the F and Cl atomic orbitals can also be the key to explain the resulted selectivity. Also, it will be interesting the kinetic behavior of polymerase assays with these isosteres to see how the enzyme efficiency can be affected. A lot of useful information in terms of base-pair interactions and enzymatic recognition can be obtained with this kind of experiment.
About Jean’s synopsis, I think that explains very well a paper that can confuse with all the numeric data showed. He understands the main message of the article in terms of explaining the questions from the X-Ray Crystallographic data. About the picture…Funny, but you can take the article’s message having a mental punch from Ali.
Groups of Wheeler, Houk and Kool did various extensive experiments (UV-Vis melting experiments, Isothermal Titration Calorimetry) in order to understand the effects that Thymine (T) substitution for the like compound, 2,4-difluorotoluene (F) has on the duplex DNA. Many conflicting evidence arise in regards with these substitutions. One evidence states the possibility for a A and F hydrogen bond formation (short distance), while other evidence states there is little to none interaction for A and F. Another disjunctive arise in regards to the favorability of that substitution for the DNA helix. Also by using Terminal Base Pairing (TBP) measurements they determined the stabilization that each base pair confers to a DNA sequence. With evidence showing that F-A pairing in the DNA is destabilizing by 3.8 kcal/mol in comparison to the normal T-A pairing. In contrast L (dichlorotoluene deoxyroboside) showed to be less destabilizing.
The article was well written and in my opinion very interesting to read. Jean’s synopsis was good and it summarized well all the key aspects of the article. I think the picture was good in the sense that it gave the article a touch of Jean’s personality.
In this article the authors set out to get to the bottom of what is apparently quite a controversial question: to what extent is 2,4-difluorotoluene (F) to able to H-bond, in its role as a structural mimic of thymine (T) in DNA. Apparently (I’m afraid I haven’t delved into this yet), F is used in studies on DNA where it is often taken to take up the same space as T (ie. not contributing steric repulsion to DNA folding), but without contributing H-bonding stabilisation to DNA folding. Unfortunately there is conflicting evidence as to whether this is a safe assumption: crystal structures have been reported at both H-bonding and greater distances, and it has been shown to be incorporated in DNA with high fidelity by polymerases. Here the authors combine both thorough experimental and theoretical work, to shed some light on the interplay of interactions taking place on substituting T with F, or chloro-analogues, and attempt to rationalise the apparently-conflicting observations.
The experimental section consists of melting point and ITC measurements of a series of different oligo-DNA strands, to determine thermodynamic parameters for melting/hybridisation, and hence the contribution to stability of different substitutions. The stands in question were chosen for minimal secondary structure (ie. undistorted double helix); 12 base-pair strands, with single substitutions, then 14 base-pair strands with two substitutions were studied, both to measure the contribution of mismatched/matched pairing; finally strands with an unmatched ‘dangling’ end were studied, to determine the contribution from stacking with adjacent bases (as opposed to H-bonding). The computational studies consisted of calculations of pairing energies of the base-pairs studied experimentally, in the gas phase and in solution.
The results taken together confirm that substitution of T for F leads to interactions with the opposite adenine base that are considerably destabilising, and that H-bonding contribution is ‘small but not zero’. The experimental results show that while these interactions are considerable, they are in part offset by a more favourable stacking to adjacent bases in the same strand. The computational results appear to corroborate an H-bonding interaction when F is constrained in pairing with A (although not sufficient to bring them together) and further dissect the interaction, suggesting that the presumption that an F-NH interaction is not strongly attractive is correct, but that a significant contribution comes from a CH-N interaction. Taken together with the previous observations of high-fidelity incorporation of F by polymerase, the lack of base-pairing stabilisation observed suggests that sterics plays a greater role in polymerase selectivity than might be imagined from the emphasis often given to Watson-Crick base pairing.
I wasn’t expecting the article to be much fun, and like Jean I found the separation of results and discussion made it feel a little dry until I reached the discussion, but once I got there I found it surprisingly palatable. Unlike the article Mariana presented last week, there were no grand claims of absolute certainty, but the combination of experiment and theory went a long way to answering the questions posed by the previous conflicting observations, and I was surprised at how well they managed to reconcile them.
I usually try and read the blog synopsis before the article being discusssed, to see if I can understand it in isolation, but unfortunately this week I didn’t get around to it. If I had read Jean’s account first, I am confident that I would have got a good sense of what was reported, and have found the results section rather easier reading. I’m not quite sure what he’s getting at with the picture, though.
Khashoor’s article emphasizes on whether or not 2,4-difluorotoluene (F) has the ability to form a hydrogen-bonded complex with adenine in DNA. They performed experimental studies of the pairing between F and related analogues in DNA. These consisted of concentration-dependent thermal melting studies, isothermal titration calorimetry and optimizations of T,F,L, and 23L with each of the natural nucleobases with the use of M06-2X DFT. They found that, although F is slightly more selective to adenine (in comparison to C, G and T), the F-A interaction remains repulsive while T-A pairing is attractive. Furthermore, the computational methods suggested that F-A pairing is 28% what the pairing is for T-A.
I have to say that I liked this article, they go into every detail when explaining their results, I think they did all the necessary experiments and that it is a very complete analysis of the matter. However, maybe they could have segmented the narrative better, since, as Jean mentions, they kind of touch up on their results over and over, which isn’t really necessary. Overall, I think it was pretty interesting.
As for Jean’s synopsis, I think it’s very well made, he summarizes all the key aspects of the article very well. I love the picture, Jean’s blog images always make me laugh. ☺
This paper is a word of caution on simplistically assuming that convenient substitutions are harmless because they seem so. The authors of the paper undergo a complete investigation of the effects of substituting the 2,4 carbonyl groups on a thymine base to fluorine atoms (F). The contradicting reports that had been made using this small substitution.
Using calorimetric experiments, the authors showed that F is quite destabilizing, even when pared with adenine, the supposed analogue to complement F. The authors make a very nice case explaining the reasons (interactions) responsible for the destabilization. Even more interesting is to see how, taking into consideration the sterics of the analogue they can reason out why the effectiveness and selectivity of the polymerase is retained while the base complementarity stabilization is lost.
Another technique that allowed them to give nice explanations to the observations was computational modeling. Some quantitative input on the interactions present in the molecules was also obtained through the computation.
Finally, their suggestion that molecular breathing accounts for the different structures determined for this analog through crystallography I found a little odd, because in crystallography, moving parts are usually blurred or simply invisible in the diffraction pattern.
The synopsis was great, and explained the paper in a clear and concise way (considering it’s a long and rather dense paper). I’m glad Jean was around to explain the picture when I first saw it (I really don’t follow boxing). He gets points for giving me a good laugh.
In this full article, Kool and Wheeler explain a recent debate about the ability of 2,4-difluororoluene (F) of hydrogen bonding with adenosine. They say that the ability of F to hydrogen bond had been reported to be very small, however x-ray crystallography studies have indicated possible bonding interactions between F and natural bases in nucleic acid sequences and DNA polymerase sites. For their studies they anaized the pairing stability and selectivity of F. They also used dichlorotoluene deoxyribose (L) as a somewhat comparison of F. They performed various studies using UV/Vis melting to see how these pairings would affect the stability of duplex DNA. The data obtained from their experiments confirmed that F-A pairing destabilizes the DNA, when compared to a T-A pairing. F also loses selectivity to A because their interactions are weaker than the interactions of A and the solvent. L showed to be actually less destabilizing than F. The computational data showed that F-A paring could be due to interactions between the CH-N and not the F-HN. Also crystallographic studies showed that the differences in the crystal structures for these pairing can “breath”, which explained the fact that they see different distances between the molecules.
This article was very interesting, and they explain very well the data that they obtained, both experimentally and computationally. I think that they performed all the necessary experiments to obtain their data. About Jean’s synopsis, it was really good and helpful because it summarizes the main parts. The picture is very funny and creative.
A very enjoyable article is presented by Prof. Kool and collaborators integrating thermodynamic experiments and computational studies to explore the possible formation of a hydrogen bonding interaction between F and A in DNA duplex, as suggested by X-Ray Crystallographic data. To accomplish this they study the pairing stability and selectivity of F and L, a fluorinated and chlorinated analogues of thymine, with adenine in DNA. Their results showed that F-A pairing in DNA is destabilizing by 3.8 kcal/mol in comparison with T-A pairing, resulting in a repulsive interaction. Also the L-A pairing was found to destabilize the duplex more than F-A. Results from the computational studies indicate that the interaction seen between F and A are due to the interaction between CH-N and not from a F-HN interaction.
As I mention earlier, I really enjoy this article, for me pairing experimental results with computational studies is always a weaning combination and in our field is the way to go. Also, for me the highlight of the article is the use of terminal base pairing measurements to isolate the energetic contribution of base stacking from the one of base pairing. I know that this technique is not new but for me is the first time I have seen it. Isolating the energetic contribution of hydrogen bonding, specially the bifurcated one, from the stacking in our system is something I always wanted to do, but that’s from the next one to come.
Jean’s synopsis was really helpful, well made and covers the essential components of the article.
In my personnel view the ‘study and discussion of hydrogen bond in DNA is always great and important’.
This is very nice article regarding the ‘measurement and theory of hydrogen bonding contribution to isosteric DNA base pairs’ published by Dr. Wheeler (Computational Chemistry, Texas A&M) and collaborators.
Substitution of thymine (T) like compound, 2,4-difluorotoluene (F) and 2,4-dichlorotoluene (L) in the sequence of a DNA duplex. In F the carbonyl groups that function as hydrogen bond acceptors in T are replaced by fluorine, resulting in a less polarized structure with similar size and shape. Some crystal structures of F substituted DNA duplexes shows that the distance between Adenosine (A) and F is relatively short, implying the possibility of hydrogen bond formation between F and A, while L shows a larger distance implying little to no interaction.
In order to understand the effects of these substitutions, the authors Wheeler and Kool have done the impressive set of experiments (performed extensive UV-Vis melting experiments, Isothermal Titration and Calorimetry on selected sequences).
In the experimental procedure it was shown that F-A pairing in the DNA is destabilizing by 3.8 kcal/mol when it is compared with T-A pairing and also they said that pi-pi interactions contribute to the stabilization of DNA duplex. These interactions measured by terminal base pairing (TBP) measurements.
From the results F destabilizes the DNA duplex, this is because hydrogen bond formation between F and A is weaker than the interactions of A with the solvent. It was also found that at the end of a duplex F-A interactions are repulsive while T-A interactions are attractive.
Computational results complement the experimental results. It was found that the interaction of F-A arises from the CH—N interaction instead of the expected F—HN interaction. L-A pairing was found to be weaker than F-A.
Finally, thank you Jean for selecting such a great worth full article and the making of synopsis is very nice.
In this article Kool and coworkers studied the interactions between Adenine and four different deoxyribosides: thymine (T), 2,4-difluorotoluene (F), 2,4-dichlorotoluene (L) and 3,4-dichlorotoluene (23L). These analogues are incorporated in 12- and 14-mer DNA and studied by: melting experiments (UV-vis), calorimetric measurements (ITC) and computational analysis. After thermodynamic data is gathered interesting relations were made to discuss the effect of electrostatic, geometry and pairing selectivity. Finally the relevance of the results were discussed from the crystal structure and DNA replication perspectives.
In my opinion this is an advanced/challenging paper with a lot of data that teaches about how to design experiments to gather important thermodynamic data.
Is there any Guanine mimic (GF)? By changing N-H1 for C-H and the amide oxygen by Fluorine an interesting isosteric analogue might be made.
I like Jeans’ cartoon not just because I am a boxing fan, but because the first message that it gives me is a competition assay between Thymine (T) and a T mimic (F) were T wins without any doubt. The synopsis was also very good, it is concise and summary the main aspects of the paper.
Always someone has to do the dirty job or I should say, the fundamental studies. No ones want to do them, but everybody wants to know what comes from them. I do apologized by starting my comment like this, but it is a reality.
I don’t want to be redundant by stating a complete summary of the article, because I think Jean did a great job in his synopsis. To be concise, the authors study the effect in the thermodynamic stability of some short strand oligo DNA sequences (12-14 bases) in were they substituted Thymine (T) by it’s isostere (molecule that mimic the shape and size of the natural base), a diflurorinated version at the 2 and 4 position (F). They tried to clarify different opinions related the distance between F and Adenine, the possibility of hydrogen bonding and the selectivity by a polymerase. They found that F destabilized the duplexes, not been able to form H-bonds and that the polymerase basically can do the DNA replication base on size complementarity instead of H-bond recognition. At first was not clear for me the de-stabilization since as we know DNA duplex formation is mainly driven by enthalpy and pi-pi interactions contribute to the process a lot. The F is more hydrophobic and I though it can contribute to the overall stability of the duplex, but I forgot that H-bonding is even a stronger non-covalent interaction and will affect the duplex stability when you take the interaction with the A, as they found with the Terminal base pairing measurements. They gave an explanation for the difference in the crystal structure measurements and presented computational studies.
Fundamental studies are the foundations of science and they always give the opportunity to learn and apply the knowledge in a broad manner. Jean did an excellent job in his synopsis; the picture was not so clear for me, then after reading some comments it made click.
In this paper by Kool and colleagues present a study of deeper understanding of the base paring. They replace thymine (T) with 2,4-difluorotoluene (F) and with dichlorotoluene (L) and examine their pairing selectivity with adenine and other nucleic bases. By this they lower the expectations of having H-bonding yet leaving the structure similarity. For this they worked on experimental and computational dimensions for full understanding. ON the experimental methods they showed that L and F were less selective than T and that they were also destabilizing the DNA, mainly because the F-A interaction is repulsive. On the computational studies they saw that the pairing energies, a lower percentage is due to the H bonding of F-HN and more due to the CH-N interactions
Personally I think the paper is well written and pictures and tables in specific were very helpful to understand the paper. That being said I need to say, maybe is my lack of knowledge on DNA chemistry, but as far as we have been taught, the pairing is strongly based on the H bonding, not simply on structure or space filling. I get the curiosity of, why would the crystal structures show these interactions as possible, but since the introduction they are aware that previous experimental data showed low selectivity on pairing F-A (very discouraging since the beginning). So they decided to study the fundamental of the pairing. They did a very good job correlating experimental and computational data, but they would have done less extensive experiments and would have gotten the same result. Or maybe I’m missing something.
Jean’s synopsis was really good with some good input on the paper and the picture is creative.