 So we will continue with the structure calculation of the nucleic acids. So we had this input parameters, coupling constants which translated into the torsion angles of the sugar geometries, then we also have some information from the energetics of the epsilon torsion angle. And then next we have the information from the NOEs, the NOEs information is in terms of the distances between the protons, the various protons inter proton distances. So this comes from the noisy spectra. So we have the input parameters, let me write here for the structure, the structure we have the sugar geometries, then you have the energetics, then you have also the NOEs, NOEs will give you the inter proton distances, which are the inter proton distances, these are the various proton in the noisy spectrum, these are derived from the noisy spectrum. And you know the peak intensity is proportional to 1 over 6th power of the distance, but we get certain distance ranges, so ranges, we do not say we get exactly identical exact distance that this is 3.5, 2.5 and so on, but we get ranges of distances and these are our input parameters. Now then what you do after this, we use this in a kind of an algorithm and that is indicated here. So you have here the distance constraints, you start with a particular model. In your particular model, the distance between 2 protons i and j is let us say Rij. Now from the noisy spectrum and you have a cross peak between i and j, from the cross peak intensity you say well this distance should be between this lower limit and this upper limit. So this is the lower limit of the distance and upper limit of the distance, I mean upper limit anyway cannot be more than 5 angstrom, this is the highest value is 5. However on the basis of the intensities, we can say that this distance should be between 2.5 and 3.5 or it can be between 3 and 4 or between 3.5 and 4.5, that kind of ranges we can give for the different peaks depending upon their intensities. These are called as distance restraints. Then what we have after that, you do a calculation to calculate the energy of the whole molecule. Energy consists of 2 components, one is the so called intrinsic energies, these are the various bond distances, bond angles, torsion angle energies and things like that. Then you have the NOE potential, you define an NOE potential which will tell you this is artificially introduced to potential and that is defined in this manner. So we have 2 components to it, this portion comes reflecting the deviation of the distance in your model from the lower bound, these are harmonic potentials here, harmonic oscillator potential functions. So deviation from the upper lower bound, take the scale, you have the force constant here, they are like a spring, force constant, this is the force constant there. And then you have a deviation from the upper bound, upper bound distance this much and you calculate this for all your distance pairs, let us say you have 1000 distances. For each one of those distances, you calculate this, 1000 distances meaning you have 1000 distance restraints there, each one of them have an upper bound and a lower bound. So for each one of those, these pairs of the distances, I calculate what is the deviation from the expected value. So you have this particular force constant which is kept there and then you define this potential function for violations of the distances for the lower bound and violations from the upper bound. If the violations are all removed, there even energy function has to be 0, therefore what you need to do on your computer, you have to keep optimizing your structure on the computer until you reach a stage that all the distance constraints defined here are satisfied. Once they are satisfied, then you have a E NOE is defined as 0 and that will be an acceptable structure for you once you have reached that stage. And this is done by what is called a particular algorithm called as distance geometry. Now distance geometry algorithm takes a certain set of distances and calculates, optimizes the structure to satisfy your distance constraints. For example, to illustrate this point to you, let us say I have a configuration which is like a triangle. If it is a triangle like this, there are three atoms in a triangle, how many distances I have here? I have one distance here, another distance there and the third distance there. So if I define the three distances, then this triangle is uniquely defined. So if I have a quadrilateral here, let us say, then I have four distances 1, 2, 3, 4. If I define the four distances, the quadrilateral is uniquely defined. So in the same manner, to define the structure of the entire molecule, I must have a certain number of distances and which will define the structure of the molecule. Therefore, you have to develop an algorithm. This algorithm is defined called as a torsion angle distance geometry. This algorithm what I am describing here is called as TAN D. This was developed in our lab by a student who called Ajay Kumar and TAN D, torsion angle based distance geometry. So you start from a particular structure here. So this is a very random structure. These are not necessarily energetically most favorable. Some kind of a random structure you have for a particular molecule. This is the 12 more DNA segments here. And then you put in the constraints and allow the molecule to optimize the structure, allow the algorithm to optimize the structure by varying the six torsion angles along the backbone and varying the sugar geometry. The sugar geometry has to satisfy the constraints as you mentioned here from the coupling constant. You can also define the sugar geometry in terms of the distances corresponding to those sugar geometries. And these are within the sugar ring, 1 prime, 2 prime, 2 double prime, etc. You can translate the sugar poker in terms of the distances as well from what you get from the coupling constants. So you put in those constraints there and then you optimize the structure in the computer so that you ultimately have to reach there. Now you see these are the various intermediate steps, how the structure is changing. Start from here, the structure is changing as optimization is going on and eventually you see it come out with a very beautiful helical structure there, helical structure. Now it is a single strand here. In this case it was a single strand. Now what you do is after that you can add constraints of the hydrogen-bound base pairs. Then you can put base pair constraints. Once you put the base pair constraints and calculate the structure then you will get the duplex structure for the molecule depends upon the sequence what you have. So therefore you will have to start with any arbitrary structure. Start with an arbitrary structure, randomized values of the torsion angles, randomized sugar geometries, these are not necessarily the most stable structure. The energetically there may be quite a bit of steric contacts etc etc, all of that may be there. Now the EF part in the sugar geometry function which I have here, so EF part which is there if there are steric contacts etc, this will eliminate those because this also has to be taken care. This particular portion involves all the steric contacts and things like that. The NOE part is taken care only in this part. So therefore but this steric contacts also had to be satisfied. So when you do this put all that together in your algorithm, here distant geometry algorithm. So you reach a structure which is a beautiful helical structure satisfying all the NMR constraints. So that is from the structure calculation. Now certain times the hydrogen-bound base pairs, base pair information can be obtained from looking at the heteronuclear NMR. Therefore you will have to use carbon-13 NMR or nitrogen-15 NMR but this will require labelling of your nuclei with carbon-13 and nitrogen-15 and this will have to be obtained by specific synthetic procedures and these are certain examples shown here. I will not go into the details of these ones. A typical example of the spectrum of a particular molecule here and you have the carbon chemical shifts in this area and the proton chemical shifts in this area and all these cross peaks which will identify, this helps you to identify those cross peaks. This is the heteronuclear correlation spectrum and this actually is extremely helpful to figure out which ones are close by and that is various experimental sequences are present here and in this case you see these carbon chemical shifts are quite distinct. Carbon chemical shifts are C2, C6, C4, C5, C6, these are very distinct. These are quite wide range of chemical shifts are present therefore you can apply very selective pulses. We are not going into the details of these pulse sequences but these establish correlations of the type here. So here for example for the you will establish correlation from this particular proton to this carbon, from this carbon to this carbon and this carbon to this carbon you will establish correlations there and these ones appear at very distinct chemical shifts and similarly from here you can establish correlation from this proton to this carbon, this carbon you can go to this carbon and so on and so forth. You will also establish correlation from this proton to this carbon. So therefore such kind of correlation experiments are present and here we show you the chemical shift ranges. The chemical shift ranges for the various basis where all do they occur. This is for anionine, gonine, thymidine, ureidine and citidine. After all of these you have the proton chemical shifts anyway which is all known from the proton spectra. This we have already seen and these are the carbon chemical shifts. The carbon chemical shifts appear very distinctly in certain region here and the C4, C5, C6 you see they are very distinct chemical shift ranges. Therefore this will allow you to apply selectively pulses to this different carbon and therefore you treat them as individual channels and the magnetization transfer can be affected from one carbon to another carbon to another carbon and so on and so forth. So this similarly you have this information for the gonines and the citidines. And the comparison between the RNA and the RNA chemical shifts that is indicated here. So you have all the proton ranges which you already discussed earlier. These are the chemical shifts for the protons and the distinct difference between the DNA and the RNA is the H2 prime chemical shift. H2 prime is shifted to a higher lower field in RNA because with oxygen present at the 2 prime position. Whereas in the DNA these ones are at 1.72 to 2.3 and this is a very unique area which will allow you to obtain the coupling constants from the structure and that is what is shown here. So the 2 prime deoxy-beta deribos this we have already seen before. So we will not go into that. Now we will switch to another kind of structure. So far we are talking about duplex structures. Now as I discussed earlier the DNA has to fold itself into multiple structures and there are various ways it can fold and various ways of hydrogen bonding are possible. One of the unique structures in this is the so called G-codorplex. We also talked about this in the hydrogen bonding patterns here. This is the G-codorplex. This G-codorplex means the G-trand. So G-trand there are 4 Gs with the hydrogen bonding pattern which is like this. Very symmetrical hydrogen bonding pattern it is present here and that is indicated in the schematic here. So you have 4 channels 4 strands which are running in a parallel way then you have these 4 Gs which are hydrogen bonded in this manner. Very symmetrically they are placed in this manner you have the hydrogen bond. This is one particular structure. But you can have many other structures. Many other ways this base pairing can be obtained. The strands need not always run parallel. So here the strands are running parallel but here you see there is a 3 plus 1 hybrid. So 3 strands are going in one direction and one strand is going in the other direction. And here you have anti-parallel 2 strands in one direction this and this are one direction this and this are in another direction. And all of this even so this 4 G-trand can be formed. What will then happen is that depending upon the orientation the glycosidic torsion angle will adjust itself. So with sometimes it can go from Nt to Sin to bring this gone in in resistor to be able to form this sort of a hydrogen bonding scheme. Now in this you have again an anti-parallel structure but now you see it is looking this way. We have this like this, this one like this and this one is like this and this one is like this. So all the 4 you can see the way the hydrogen bonding parallel is done. Different ways you can form the anti-parallel structures. And in this lower one here now you can have a loop. So double chain reversal loop chain runs like this there are only 2 G-trands. Chain runs like this then it actually takes a long loop and comes back and goes in a parallel way here. What is present in this loop area? They are not G's they may be some other nucleotides like the T's or the C's and things like that when they are present the chain can run like this double chain then there is a loop and these are put it in place there and then it can form a tetrad like this. Now similarly this can form within instead of going like this now if these are going parallel this is the turn it has to take. But if they are anti-parallel if these 2 chains are anti-parallel then you can simply go in a loop like this and form a strand like this and form a pair here. So similarly you can have another one here this across the diagonal so to say. So it goes from here to here forms then it folds the diagonal loop. So these are the this is called the edge wise loop this is the double chain reversal loop. Then in this situation you have one molecule which folds in different ways to form a structure. So in this case you see the chain starts from here it starts from this point it goes down then the loops then comes here then it goes in the opposite direction then loops comes to this position then it goes direction then this chain this kind of a chain loop and then you have this one. Therefore goins come in resistor to form a structure of this type. Now here it is 2 simple loops like this so the chain starts here and then the loop here goes in this direction then there are 2 molecules here there are 2 the connection between these 2 is not shown. So then you have another one going here and going like this and these ones are held together in this parallel fashion. So you see there is enormity there is so much variation possible in the structures and this is why it is not surprising that the entire DNA can be packed inside a small nucleus forming different kinds of structures. Now it is of course at the same time it is also a challenge to figure out what sort of a looping is happened. This has to be done from the proper NOE based experiments and the heteronuclear experiments to establish the nature of the hydrogen bonds which hydrogen bonds which an hydrogen is involved in the hydrogen bond. So all that has to be seen from this sort of different kinds of experiments. So here is one illustration there the illustration of course we will not go into the details of this what is the nucleotide numbering etc here how these were obtained but this is showing look at this the chemical shift spread this is the record this experiment is recorded in D2O this is recorded in H2O and all of these peaks are present here okay even D2O some of these are present these are these are the immunoprodoms the immunoprodom region the quadruplex the immunoprodoms appear from the 10.5 ppm to 12 ppm these are all G immunos you see how many how many are there these are 12 here the 4 4 4 into 3 12 and all of these are distinct these are distinct there is no symmetry here because of that you are seeing all the 4 G's are a distinct and they are all present here okay you can count here 1 2 3 okay there are 12 3 here 4 here 7 plus 5 12 all the 5 G's all the 12 G's are distinct these are all found within the same molecule you have a particular sequence running like this this goes like this then you have a loop of this type then it comes down here like this then you have make another loop like this and then of course it goes turns here then another loop here and then ends up here and this so it is amazing amazing the way the chain is folding here and because of that you will have different orientations of the glycosidic torsion angle the sugar geometries will change and they clearly because of that the nonic all of the become non equivalent and you see the distinct chemical shoots for the 12 G's which are present in the G quadruplex once we have this then you will also have the NOE is between these various immunoprotons to the sugar rings and then from then you will figure out what are the sugar geometries and the glycosidic torsion angles see what are the individual glycosidic torsion angles okay now this is another example there how these various the nucleotides are formed there are 2 nucleotides one particular sequence which goes like this other one which is like this then you form a quadruplex here and in this case this is a different kind of a structure you have this one go this starts here goes like this and comes down and another one which goes like this and comes out these are 2 parallel loops these are going in the parallel they are on the same side these 2 loops are on the same side of the quadruplex of the structure here the 2 loops are on the opposite side of the structure both the structures are possible both the things may coexist so when you have this you will have peak coming from both of them okay there is a certain symmetry here and those peaks this and you will establish the connections from the base base NOEs base base NOEs here and and the sequence that is given here is this T g4 T 2 g4 T these are the 2 4 G's which are forming the quadruplexes here okay so these are 2 3 4 5 then you have 2 3 4 5 8 9 10 11 because you see then you have the T 2 here the T 2 is in the loop and in the result T 2 is in the loop G4 T again this 4 genocletides here these are 8 9 10 11 and in the same sequence can form a structure of quadruplex in this case this is the symmetric this is symmetric and you have both the structures possible and you will see separate peaks for each of these structures and therefore you see so many peaks beautiful spectrum you can see all of these are very well resolved here all the amino protons the red and the things you can see for all the different strain peaks can be identified they are identified with the different colors as I indicated in this so therefore this can actually become as complex as protein spectra so see the small molecules even a small molecule like this is presenting such a wonderful dispersion and so many peaks and it is of course quite a challenge to establish all of these connectivity okay now this is the simple example of a small molecule a small molecule of T AG AG 3 T this is only a timer okay in potassium solution in potassium condition incidentally notice that this will not form in the in sodium the quadruplexes are formed in the potassium solution because it so happens that the space between in the G tetrides the potassium can fit in very well and that will stabilize the quadruplex structures the sodium will not be able to do this therefore typically you do not find this in the sodium solutions we will find this in the potassium solutions as in quite an interesting feature and in this case look at the stability of this quadruplex there are G3 there are T 3 G tetrides there and this is the immunoprodons of the G's okay and these are of course the aminos here and the base protons H8 H2 and the immunoprodons are stable all the way out to 330 degree these brought out here melting if you see this is extremely stable even your normal duplex DNA of the 12 more DNA will not be stable you will not be so much stable typically you will have the melting temperatures of 45 degrees 50 degrees and things like that so this is quite stable structure and these and by looking at these of course you can identify which nucleotide is which and incidentally in this case we also discovered something unique and that is this particular slide we show you you see here a cross peak these are the sequential connectivity as we see in the normal case this is the parallel standard four-stranded structure the G quadruplex you have the identification of the sequential connectivity is here T5 to G4 G4 to G3 G3 to G2 G2 to A1 and incidentally here you see a cross peak to the A1 H2 and this cross peak is seen to what this is seen to the amino protons these are to the amino protons this is A and H2 amino protons are normally not seen and they are typically in the in this area chemical shift area amino protons are not seen because they exchange with water unless they get involved in the hydrogen bond but now you see here we see an interestingly in this particular sequence AG3T you see these amino protons and what are these these are a two different mixing times this is 180 millisecond mixing time of the nosy this is 60 millisecond time nosy and you see these are present why did we have to do these at two mixing times because they should not be spin diffusion somebody may argue that this is not a direct interaction but there is a spin diffusion but when you go to small mixing time there is no spin diffusion direct and you see amino proton NOE is to the A1 H2P normally you do not see this for example if you took this molecule this is TAG3T TAG3T you do not find that and what does this tell you this actually is telling us that the A is also forming quadruplex A is also forming a tetran and that was an interesting observation here and of course this was confirmed by various other amino NOEs here from the amino protons you are seeing NOEs to the various amino protons amino protons to amino protons you see NOEs and also to these amino protons and otherwise you normally do not see the amino protons but here you are seeing the amino proton peaks as well because these ones got stabilized in the formation of a tetrad and that is shown here therefore this was a tetrad we called this as an a tetrad you can see the hydrogen bonding scheme here bond here see see this is the hydrogen bond now what happens is this amino group is close to this proton here and this is the H8 proton that was the peak which you were seeing H8 proton and this amino is also close to this proton this proton is H2 proton and we were seeing this amino to the H8 amino to the H2 we are seeing this and now there are two ways one can form this hydrogen bonds and these are the two different ways we have shown here and that indicated the structure one could calculate the structure now on the top here you have this 8 tetrad you not only have the g tetrads here there are 3 g tetrads there and the top you have the 8 tetrad as well and that was the previous one which I showed you in this one this is the 8 tetrad 8 tetrad is formed in this manner the hydrogen bonding scheme is slightly different in these two cases and these are called as I will show you what these ones are these are three two different kinds of structures as stacking of the A1 this shows the stacking of the 8 tetrads over the g tetrad this provides the stability to the 8 tetrad so the one which is in cyan color is the 8 tetrad and what is below is the g tetrad so look at this how this stacking is happening this stacking of the basis the purine basis provides enormous stability to the structure of the 8 tetrad otherwise you normally do not see this okay now this is another discovery there this is called the t tetrad so okay once you have the g tetrad can you also have a t tetrad indeed it turns out that you can also form a d tetrad and that is evident from the t4 NH to the t methyl so this NOE is very characteristic of that one and we will not go into the details of this discussion there with regard to the analysis this is typically the standard analysis of the one prime region of the base to the one prime area and then to the base to the two prime area two double prime area base to the methyl area and this one is the amino protons the amino protons to immunoprodons to the various NOEs immunoprodons to the various Gs there and then immunoprodons to the t4 NH okay now this actually clinched the issue that okay there can be t tetrad as well the t tetrad is also formed and that is shown in this particular way the t4 NH to this one you see is seen even at a high temperature like 313 these are the typical from the g tetrad this four Gs which are present here okay these form are shown here and one of these t is the central t is also involved in the tetrad formation and that is what you are seeing in this area there okay this physical peak is shown okay now t tetrad there are two different ways the hydrogen bondings can be formed which oxygen is involved in the hydrogen bond okay now this is the oxygen and this is the NH this is the t t NH and this is hydrogen bonding like this to the oxygen this uses the O4 this is the O4 in this case this is the O2 because two carbonyl groups in the thymine right at position 2 and at position 4 so you can use either one of those to pair with the t3 NH so once we have that then you have this kind of a two types of hydrogen bonding schemes and you can have different structures depending upon that you see you can see different kinds of structures that can be formed depending on this is the stereo picture of the one particular structure and you can have this is a space filling model of the same structure and here you have the comparison of a this kind of a tetrad G there are 4 Gs continuously opening here and in this case the 4 Gs are interspaced by a t in this case okay now what is the result of that you can see there is a kind of a flattening of the backbone here in this structure and that is this one and in this case this is more straight the backbone is going in the straight and the tetrad is formed in the middle it is the G TGG TGG C you also have the T tetrad in the middle okay so this sort of structures are formed and okay then I think we have to investigate this various kinds of DNA structures depending upon the sequence what we have the variety of structures is quite large you have we have talked about the quarter flexes there can also be triplexes the triplexes we talked about the triplexes earlier in the previous lecture when you talk about the structures and there are also characteristic NOEs for the triplex structures as well depending upon the base pairing scheme whether it is TAT or AAT triples we will have different kinds of NOEs so with that we will be able to calculate this triplex structures as well so therefore this establishes the enormity of the DNA structures which certainly enables the packing of the DNA inside the nucleus so I think we can stop there and that completes the nucleic acid structure calculations and the varieties of nucleic acid structures.