 So, we have been discussing about the various aspects of nucleic acid structure, how the whole thing started with fiber diffraction, single crystal X-ray data on shorter oligonucleotides has helped in obtaining great details about the structure of the nucleic acids at atomic resolution. The various aspects of the structure we have discussed and we mentioned about the different types of models, the BDNA, ADNA, CDNA, DDNA and things like that, we also indicated the importance of this nucleic acid with regard to the hereditary functions and we also talked about one of the new discoveries which is called as the GDNA and here actually this slide is a continuation of that discussion and makes a direct comparison of the most prominent nucleic acid structure which is the BDNA and then we have the left-handed which is the GDNA and this is the comparison here of the two. By large you can see here this BDNA which is the one which is prominent one in all systems, it is a right-handed helix, you can see the helix goes in this manner and there is a duplex. Of course, the other strand also goes in this manner here, both are right-handed helices but they run in opposite directions, the 5 prime end to 3 prime end one sense, other one goes from the other end 5 prime end to 3 prime end and they are held together by the base pairs here and these base pairs are called the Watson-Krieg base pairs. So these are all the base pairs and it is the overall features of the BDNA are given here. So you can see there is a big groove which is indicated by this here, this is the big groove, it is called the major groove and there is a smaller groove which is called the minor groove here and therefore you can see that all the base pairs one end opens in the minor groove, the other end opens on the major groove. So these are the characteristic appearances of the BDNA. On the other hand the GDNA is actually left-handed helix, you see the helix goes like this and it is also zigzag, it is not as smooth as it is as the BDNA is. It also goes in a zigzag manner and you have the left-handed helix as indicated by this and there is a deep, there is a deep groove here and there is a bulge at this point. So therefore this structure is not as smooth and symmetric as this one is and here are the parameters which are given for these two structure types. This is the firstly right-handed DNA, BDNA is right-handed versus a left-handed DNA here and the number of nucleotides per turn, so when the system repeats itself, the cycle repeats itself, how many nucleotide base pairs are there? The BDNA has 10 base pairs and therefore we say 3.4 angstroms into 10, there is a 34 angstroms is the rise per, that is indicated here next and then whereas the GDNA has 12 base pairs per turn and the diameter of this helix, the entire helix, here this is 20 angstroms and here it is 18 angstroms from what less and the rise per base pair is 3.4 angstroms here, this is 3.7 angstroms here and the total helix pitch that is 34 angstroms because if you take 30, 10 base pairs per turn 3.4 into 10 that makes it 34 whereas this one goes to 45 angstroms, one particular turn what is the rise? What is the rise per one turn, 45 angstroms here and the random per base pair there is a rotation per base where this is 36 degrees and this is minus 30, what is minus 30 degrees because it has to go in the negative sense because it is a left handed DNA. If I take the right handed rotation as positive, the left handed rotation will be negative and therefore it is minus 30 because 36 into 10 will be 360 degrees that makes a complete turn all the way and minus 30 into 12 makes it 360 degrees and therefore that is 12 into this will be minus 30. And there are grooves here, wide and shallow through different kinds of goals and these are very well stacked and these the base pairs are not very well stacked in the ZDNA. So you see the stacking is not very good whereas here the base pairs are parallel to each other and they stack one over the other and that is a quite a good structure, symmetrical structure. And now there is one more important parameter which is not of course visible from here that is called as the glycosidic torsion angle. What is the glycosidic torsion angle, I will show you that so on. And then I also mentioned in the last time that in the case of BDNA it is a monomer which is the repeating unit, monomer is the repeating unit whereas in the ZDNA it is the dimer which is the repeating unit and that is why these are given separately here. And for the BDNA there are two kinds of glycosidic torsion for the ZDNA, two kinds of torsion angles are indicated here syn and Nt. One of the nucleotides the guanine nucleotide has the syn conformation whereas the citidine nucleotide has the Nt conformation. And the sugar geometries whereas in the BDNA they are all C2 prime endo which is uniform whereas here it alternates. So C3 prime endo guanine syn has the C3 prime endo sugar geometry and the cytosine has the C2 prime endo sugar geometry. Therefore this makes the dinucleotide as the repeating unit. Now what is the glycosidic torsion angle, this is explicitly indicated here. So you see here this is the sugar ring, the sugar ring is a 5 membered ring here. This is shown for the RNA but it does not matter it is the same for the DNA as well. So in the case of DNA this OH group is ribless by the hydrogen same here. Now the glycosidic torsion angle is this. This is the glycosidic torsion angle, rotation around this bond. This is labeled as chi and a proper model of that this is the schematic on the left hand side and the proper model of that is shown on the right hand side. So here it is both all the things are included in this. So this torsion angle you notice here what is the difference between these two. Here it is shown for the purine ring, this is a purine base and this one is the guanine what is chosen here is a guanine because you have an NH2 group here and adenine also has an NH2 group here. So this is guanine has NH2 group here adenine has an NH2 group there. So if the rotation is around this bond what is the difference between these two? See the 5-membered ring this is the 5-membered ring of this purine ring comes on the side of the oxygen here. This torsion angle is defined with respect to these four atoms if it you can either define this way or you can define with respect to these four atoms so either way it is the same thing. So you can choose the convention where you want how you want to choose one particular convention is used. So the essential point to notice from the structure point of view is the 5-membered ring here comes on the side of the oxygen and here there is a proton attached notice the proton is not shown here but there is a proton here there is a proton here and that proton is called as the H8 proton and in this case this proton is here. So the protons are on two opposite sides in the two cases. Now in this case this is called as the thin conformation whereas this one goes outside compared to the relative to this oxygen position and in this case it comes closer and this is called as the anti conformation. Typically it is shown in terms of the structure when you actually build a model how does it look? So you have the C2 prime endo geometry here sugar ring is in the C2 prime endo geometry and the anti glyco static torsion angle. So this orients rotation around this bond is indicated here you see this NH2 groups comes close and this is the proton this is the indian this is the proton here. See this proton comes closer comes closer to the oxygen of the sugar ring here so whereas that proton is far away this far away compared to the oxygen that is this one here this is far away as I mentioned this is far away but this one will now be this will be closer to the sugar ring sugar ring protons here the H1 prime protons that will be close this proton will be close to the sugar ring protons here whereas this proton will will be further away from the H1 prime or the H2 double prime protons in the case of the Nt conformation, C2 prime end of sugar and the Nt position of the glycozolic of torsion angle. So and this will be important, why I am mentioning this is because this will be extremely important when we actually use these information for structure calculations with regard to determination of the structures, what are the short distances from the NMR point of view which are the important distances one has to see, from that point of view this is very important. That is why I am pointing out these things at definition of the glycozolic torsion angle. So we have C3 parameter geometry for the sugar ring in A type structures or RNA structures and B type structures it is the C2 parameter sugar geometry and the glycozolic torsion angle is Nt conformation, it is both in BDN and ADNA whereas you have possibilities of glycozolic torsion angle in the SIN and Nt conformation in the case of GDNA and there are other possibilities as well. Now is that all about the nucleic acid structure, surely this is not why we are saying this because you remember the DNA if it were duplex as indicated if are so long the length of the DNA, length of DNA will be of the order of centimeters, considering 1 billion base pairs and DNA length in this every cell it will be in the order of centimeters because 34 angstroms per rise and 1 billion base pairs if you take it will come to the order of centimeters. Now this cannot be accommodated in a cell which is only of 1 micron size and the nucleus is even smaller than that therefore DNA folds, DNA folds multiple times there may be stretches of various other kinds of strands where other kinds of base pairing possibilities there may be single strand possibilities all sorts of things can be present and in the case of RNA we mentioned already that the mRNA is a single stranded RNA and in the case of tRNA and ribosomal RNAs there can be different kinds of structures possible with various kinds of folds of the backbone and resulting in various different kinds of base-base interactions. What are the possible base-base interactions that is what we are going to see here. So you can have so far we talked about the purine pyrimidine base pairs that is the one which is indicated here on the top, At base pair now this is purine pyrimidine base pairs. So what is this is the pyrimidine ring and this is the purine ring this is AU and GC for the AU how many hydrogen bonds are there 2 hydrogen bonds and for the GC or the for the GC we have 3 hydrogen bonds which are holding them together. Notice clearly which what is the nature of the hydrogen bond this particular position is hydrogen bonded to this nitrogen here and what is the R, R is a place where the sugar ring is attached. Here the sugar ring is attached in this position and in this case in the purine sugar ring is attached at this position this position is called as the N9 position. This is N9 position and this is N1 position this is N1 nomenclature goes in that manner whereas for the pyrimidine this is this position is called as the N3 position N3 the numbering goes in that manner. So the standard nomenclature with regard to the IUPAC conventions etc this is the N9 position this is the N1 position this is the N3 position this is all we have to remember here. So far as the purine pyrimidine base pairs are concerned these are the Watson Crick base pairs which are there in this BDNA. So duplex go the backbone is in green and purple are the base pairs this is the possibility. Now there is another possibility also here see look what is involved here so what has happened here this base pair this is different from this base pair the reverse Watson Crick this portion is the same the A is in the same configuration NH. Now what has changed in this NH NHN the R is here whereas the R is here in this case the R is up which means there is a rotation with respect to that with respect to this axis with respect to this axis there is a 180 degrees rotation therefore this R has come on to the top this R is at the same place the sugar ring is at the same place here the sugar ring goes on the top. So therefore this is called as reverse Watson Crick and same happens here as well nR is here and in this case the nR is there. So this is both in the AU as well as the GC base pairs it goes in this manner. So this is the other possibility of hydrogen bonding surely these people are toyed around with all these possibilities finally came up with this it turned out that this what they came out was finally the correct one with regard to the BDNA. But this kind of a things also people have toyed around to see whether this one fits some experimental data did the building model model building did not fit into this once they came out with this but this can be there this can be there in other kinds of situations where the DNA folds and various kinds of interactions and happen these ones can be there and then look at this this is called as the reverse this is the AU Hooke-Steen this is the Hooke-Steen base pairing Hooke-Steen base pairing what happens here the NH is base pair to this the this configuration is the same here. Now this is the purine ring and it is this nitrogen it is this nitrogen the five member ring nitrogen is involved in the hydrogen bonding with this NH here whereas in this case it is the NH of the N of the six member ring whereas here is the N of the five member ring so that is here this is free here right this N is free and that now comes here to take part in the so therefore there is a rotation of the glycoesthetic torsion angle which brings this nitrogen closer to this hydrogen here this is the N3 of U is base to N7 of the purine and that is called as the Hooke-Steen base pairing. So Hooke-Steen probably which is the one who proposed this similarly there is a reverse Hooke-Steen base pair in this case the NR here NR is here NR here they are on the same side of the duplex or the on the base pair that is they are both open in the same group whereas here you see the NR is here and this NR is here and therefore this is the AU reverse Hooke-Steen base pair okay once again this is the N7 N3 but the glycoesthetic torsion angle is different and therefore this one has gone on to the other side U reverse Hooke-Steen and this one is a wobble base pair now then you have the so called wobble well so that was the reverse Hooke-Steen now this is the wobble base pair wobble base pair GU wobble see so far we talked about the AU base pair but here it is a GU now the GU is not the thing which is normally supposed to happen we used to have to have a GC base pair okay but here we are now talking about GU base pair the G pairs with U conventionally we are 80 base pair or AU base pair but here it is the G G pairs with you this also is possible and now this hydrogen bonding scheme here is like this so this N1 of G pairs with this oxygen here and the once again this is the normal GU base pair okay NR here NR here and the reverse is again the NR goes on the other side so it depends on which oxygen is involved in the hydrogen bonding whether it is this oxygen or this oxygen that is what determines whether it is in their normal way or it is in the reverse way so both this is called as the wobble base pair because this is normally not there in the duplex DNA and then surely you have the AC you see here this is the AC reverse Hooke-Steen AC reverse who will wobble and AC reverse Hooke-Steen AC reverse meaning what we normally have 80 base pair right so when the T is replaced by C we call it as a wobble AC is a wobble base pair but this is also possible so such hydrogen bondings are also possible so you have this NH and here NHN both are NHN hydrogen bonds in this case okay in all of these cases one is these are NHO hydrogen bonds in this case in these ones we have NHN hydrogen bonds one NHO hydrogen bond in all of these one NHN one NHO here also one NHN and one NHO whereas here you see AC reverse in this GU wobble we both hydrogen bonds are NHO and the reverse also both are NHO hydrogen bonds and in this AC reverse they are both NHN hydrogen bonds NHN hydrogen bonds is the amino proton is participating in the hydrogen bonds therefore you see how many different possibilities are there for hydrogen bond formation now then we can also home your purine base pairs just as we talked about purine pyrimidine base pairs you can also have purine purine base pairs so here it is a a and a a a base pair okay and different types so a a N1 okay N1 to amino so you have the one of the proton is is the amino and the other one is the nitrogen okay so you have here this amino of this is hydrogen bonded to this nitrogen here of the six-membered ring and similarly six-membered ring of this is paired to this nitrogen here a okay this is one a a N1 amino symmetric base pair and then you have a N7 N7 is involved in this case you see it is a N7 which is hydrogen bond to the amino this is the N7 N7 is hydrogen bond to the amino NH proton here and correspondingly for this one N7 is paired to the hydrogen bond here so this is we have the amino protons participating in this and similarly here as well a a N1 amino N7 amino so here you use both N7 both places N7 here we use both places N1 and here we use N1 and N7 one case it is N7 other case it is N1 so it is enormous possibilities of hydrogen bonding schemes are possible and of course only when of course you cannot remember this when you but suddenly when you actually have to build models try and understand the structures which you actually observe then one should take in account all of these possibilities how does one determine all of this this of course one determines by NMR by looking at which are the ones which are hydrogen bonded which are the protons which are involved in the hydrogen bond whether it is the amino protons amino protons which an hydrogen is involved so all of these can be determined by from NMR data so this is the G1 N1 carbonyl symmetric CG GG so you have G1 G1 is this okay now G1 is hydrogen bonded to the carbonyl carbonyl here earlier we are using NN hydrogen bonds so here is NHO so NHO hydrogen bond here okay N1 position N1 position of this goes to the oxygen of this N1 position of this one goes to the oxygen of this and both here in this case only the six member drink is involved and what happens here now this is GG N3 amino symmetric okay now here it is NHN hydrogen bonds and this is amino to N3 position this is the N3 position in this case this was used N1 position here we are using the N3 position and N3 to this amino proton okay GG N3 amino symmetric hydrogen bond and the last one is GG N7 N7 N1 carbonyl amino so N7 that is that is this one this is the NHN hydrogen bond here to the N1 and the amino is hydrogen bonded to the oxygen here so you have here one NHO hydrogen bond and one NHN hydrogen bond similarly GG N1 carbonyl N7 hydrogen bond so N1 is where this is the N1 N1 is is bonded to the oxygen here this is N1 is go to the oxygen here and this N7 is going to the amino so this is the N1 there is the amino proton on the G this G amino amino is hydrogen bonded to the oxygen whereas this nitrogen is hydrogen bonded to the amino of the G so these are GG base pairs so you can have so many different kinds of base pairs home your purine base pairs we saw AA base pairs and then we have the GG base pairs different possibilities of hydrogen bonds then hetero purine hetero purine base pairing so far we looked at AA or GG but you can also have GA base pairing so GA base pairing is indicated here different possibilities of GA pairing here so the G here you see N1 N1 N1 carbonyl amino so it is amino of the A is hydrogen bonded to the carbonyl of the G and the amino of G is hydrogen bonded to the nitrogen of A so this is NHN hydrogen bond and this is the NHO hydrogen bond so this was GA N1 N1 carbonyl amino okay so these are both N1 positions there N1 N1 okay and these are GA this is A notice here in the case of G there is a proton here I indicated to you earlier okay and what is present in the case of A there is a proton at in the case of G there is an amino here there is NH2 group this is position number 2 at 2 position there is the amino group here in the case of A at position number 2 there is also a proton okay and you have an amino group at this position there okay so this is the proton here as N2 position this is N9 and this is 8 so there is a H8 proton here and the H2 proton here okay let me write that here this is H8 and this is H2 and this is H8 so these are the positions according to the labeling and that is what we have there okay so in the same manner you have the GA N3 amino amino N1 GA N3 so where is G this is the G and N3 is this and this is going to the amino of the A so this is the NH2 so this NHN hydrogen bond is there and this amino this is at the 2 position these hydrogen bonded to the N3 of this okay and so therefore you can NHN these are both NHN hydrogen bonds here one NHO and one NHN and involves amino groups and the amino protons and the next one is AG N7 N1 amino carbon so this is the GA1 one possibility and this is the next possibility here so this amino is going to the oxygen of the A amino is going to the oxygen of the G here amino is going to the oxygen of the G okay and then this NHN and this is the N1 position is going to the N7 here N1 to N7 AG okay N7 N1 this is A this is N7 this is N1 and AG N7 amino N3 and look at this possibilities because there are so many hydrogen bond acceptors and donors in the base structure all these acceptors and donors can accept a hydrogen bond and give a hydrogen bond give a proton and that is why you have this so many different kinds of possibilities of hydrogen bonds okay pyrimidine-pyrimidine base pairing as well likewise so far we looked at the pyrimidine-pyrimidine base pairs you have possibilities of CC amino CC symmetric base pairing CC carbonyl amino symmetric base pairing and CU there is a CU pairing possibility or low N3 N3 here then UU pairing carbonyl N3 to symmetric and we will not describe it in detail further so just the main structures are shown here okay then you have possibility of UC N3 N3 and UU hydrogen bonding possible so you have so many different possibilities of hydrogen bonding and though because all of these nitrogens and the carbonyl oxygens they are all acceptors and the you have the donors are the amin amino protons the NH2 and the NH protons and these are present on both the all the bases you have such kind of acceptors and donors and that is why you get different kinds of hydrogen bonds okay now as a result of all of these now I indicated here what are the different possibilities of the structures we said so far we talked about the various kinds of base pairing schemes and where do they occur where do they occur okay now you have possibilities of a duplex here standard duplex then you can have when the running chain is going on depending upon the nature of the sequence which is present here it can assume different kinds of secondary structures single standard region here as a single standard region and you have a loop here okay there are loops coming out here in this place okay and these are the so single standard regions you will have in this this is not paired at all so this portion is paired this is not paired so all kinds of folding schemes can occur okay so these are and then you can have the hair pins so the thing go like this this looks like a hairpin right so therefore and then you have this bulges here in a duplex in between the base sequence is such that it is not pairing possibility here this fellow bulges out so you will have a bulge here okay so similarly there is a small bulge here there is only one but this is several several bases are involved in this therefore becomes a longer loop and this is only one base which is going out here such kind of possibilities are there and now in the internal loops within that within the duplex itself you can have certain regions where there are bulges in the middle so because these are not complementary why does it happen because these are not complementary sequences when there are not complementary sequence when the possibility of a base pair does not exist then you will have these kinds of loops okay symmetric loops in the parallel structures and you can have a antisymmetric structures antisymmetric loops and you have these kinds of possibilities and you can have four stranded structures so in this place so you have the three stem this structure has three stem this structure has three stem okay and we will see that such cases do occur in RNA, ribosomal RNA, tRNA and things like that and there is an actually a region which is bulged out here so the chain has to run like this so these are all the possibilities indicating the folds of the nucleic acid structure and you see here is a four stem structure and such kind of structures do occur in the functional aspects whenever there is a kind of a recombinant process going on inside the in the replication process or such kind of this kind of structures do happen in this this is a four stem process so the stable DNA structure RNA structure is one thing but the during the when there are activities going on the DNA has to open up and interact with other systems other DNA segments or other protein segments and such kind of transient structures do happen and these transient structures the for example one of them is called the holiday junction so they have here this is the four stranded structure so there are many such kind of a structure which are possible these are functionally relevant the DNA has to open itself to express express itself to form either for producing the proteins or replication process and things like that and in all those processes such kind of transient structures do occur ok now so far we talked about two bases interacting with each other but you can also base triples base triple means so three base pairs three bases are interacting with one another see here you have the normal Watson Crick 80 base pair normal Watson Crick 80 base pair and here the normal Watson Crick GC base pair and on the top comes here a third base which is the T so you have a T 80 triplet this is called as a base triple T 80 triple and here it is a CGC triple ok and look here in in this base pairing what happens in the the free position which is here the n7 position is used up for this base pairing in the for the third base ok the oxygen of this uses this amino proton in this proton here ok amino proton and the n7 is used up for the T and this place so this kind of a this triple base pairing is possible similarly for the GC GC also same thing happens the free donors and the acceptors in the Watson Crick base pair can accommodate another base in the major group if it comes on this other side it is a major group so on this side it is a minor group so you can have this sort of possibilities here so you have a GC and the G coming here ok what you add here T 80 T is the pyramid 80 and then of course the third base is the pyramid in here there is a third base which is the purine the G is coming and similarly in this case you have a 80 pair and then the a is coming the third base is a and what are these arrows here these arrows are indicating which are the short distances because these are the observables in NMR spectra see these proton what are indicated are the proton proton distances so all these proton proton distances are the ones which are short distances and we use these kind of short distances to identify or assign the individual basis and the individual protons that is why these are indicated here indicated by double-headed arrows that we can actually observe these ones by recording spectra in water now these are base triples now you can also have quadruples ok it is called the G tetrad so 4 Gs can hydrogen bond with each other and to form what is called as the G G T G G G tetrad there are 4 Gs here very symmetric see and all the all the donor and the acceptor sites are used up here so it uses this nitrogen this oxygen this nitrogen amino proton and this amino proton so all of these are involved in the hydrogen bonding in a very symmetrical manner so this produces a G tetrad which is an extremely stable structure very stable structure we will say I will show you examples of this how this four stranded structure is possible and how this can be extremely stable ok so typically how does the BSA DNA triple helix look like so you have the green one is a normal duplex normal duplex and the triple helix comes the third strand comes in this major groove ok it comes on this and then you see the third strand is hydrogen bonded to the base pairs in the duplex and this is the space filling model of the same sort of a structure and these ones are such kind of structures are possible in the quadruplexes so the four stranded structures are possible here so you have the G tetrad and you can have quadruplexes of various types various types of quadruplexes are possible these are called parallel stranded quadruplexes and here you have two strands going in one direction two strands going in other direction and you can also have loops here ok the chain runs like this and loops around come here the other chain runs like this and loops around and comes down here and these four G's which are in the thing they can form hydrogen bonded structures and in all of these is the glycothalic torsion angles becomes important ok. So and depending upon what is the orientation of this you can have different kinds of base pairing schemes and so that determines the base pairs then the last one structure which has been discovered sometime back is called the i-motif the i-motif is a structure which is between the cc plus see the cc base pairing is possible but the one of the C gets protonated here and because of that protonation it gets a positive charge therefore it is called as the cc structure. Now here what happens is you have two duplexes two duplexes interdigitating that is why it is called as i-motif ok. So it is it can be different molecules or the same molecule loops around turns around and things like that and comes back and that is shown is in this case the chain starts here let us say goes like this goes like this and then goes like this and then turns around and then see these are the various possibilities that is all these are required because of the DNA has to fold in many different ways and all those structures will have to be stabilized this stabilization happens because of the different possibilities of hydrogen bonding pairs. So this is actually a quarterplex how does the quarterplex structure look like so this shows here the four how are the four strands accommodated in this ok so that is the quarterplex structure you have this four colors here these are the individual strands which are going as you can form a structure of this type this is the DNA quarterplex. So I think we possibly stop here and now of course here the DNA protein complexes can be formed DNA protein complexes are important in various now here it shows how a duplex DNA can fold and accommodate the proteins and the proteins can interact with the DNA and proteins will have to express and protein will have to interact at various sites in the DNA duplex or with other various kinds of structure that are possible here with the single standard areas the double standard areas different possibilities of interactions are possible. So there is a whole variety of structural possibilities enormous complexities in the DNA structure and people thought the only duplex DNA is a simple thing that is the only thing about the DNA but of late we know that there are so there is so much more about the nucleic acid structure which needs remains to be explored much more needs to be done with regard to the nucleic acid and especially RNA structures so many different varieties of RNA structures are possible. So I think with that I will stop here.