(The reference numbers cited in this summary refers to the numbers in the list of publications)
Ref # 1-12: Publications from the Ph.D work
Ref # 13-26: Postdoctoral work
Ref # 27 - : Independent work, in which JC is the senior author.
During the independent scientific career (i.e. from 1979), I
have tackled ten fundamentally important contributions on the
synthesis, structure and function of DNA and RNA and their components
using the combined techniques of synthetic chemistry, physical organic
chemistry, conformational analysis by high-field NMR and molecular
modelling techniques (they are all appropriately marked in the list
This understanding by 15N-NMR spectroscopy showed that the monitoring of the electronic structure in nucleic acid components can lead to the foundation of appropriate choice of protecting group particularly for guanine for cleaner synthesis of oligo-DNA and oligo-RNA (For example, N1 protection leads to the enhancement of nucleophilicity of N7, whereas the O6 protection by aryl based group reduces the N7 nuclephilicity more than alkyl based group).
Hence, we are one of the first to synthesize some model lariat-RNAs [116, 121, 131, 137, 139, 144, 145, 157, 158, 167, 188, 190, 193, 206] to understand the uniqueness of their structural make-up. This we examined by high-field NMR [150, 151, 153, 162, 163, 170, 172, 176, 183, 185, 198, 202, 209, 218, 225, 226, 227, 240, 241] to understand why are they formed in Nature as a prerequisite for the ligation of exons, and why Nature has chosen to preserve some RNA sequence elements in the branching junction of the lariat-RNAs? This study showed that: (i) adenosine at the branch-point has a completely different tertiary structure than the counterparts with guanosine, cytidine or uridine as the branch-point nucleotide, and (ii) the competing 2',5' and 3',5' stacking at the branch-point acts as an 'energy pump' since these two differently stacked states have different free-energies associated with their conformations. Thus a transition of the 2',5' stacked conformation in a branched RNA or lariat-RNA to the 3',5' stacked conformation releases the free-energy of activation, which drives the transesterification reaction in the splicing.
We for the first time showed [218, 225, 240] that some of these synthetic lariat RNAs undergo unique self-cleavage reaction (k = 0.16 x 10-3 min-1) which are the biomimetic models for the studies of the ubiquitous self-cleavage reaction (k = 0.5 min-1) in the catalytic RNA found in natural ribozyme-RNA substrate complexes such as in hammerhead, hairpin and delta ribozyme and in Neuropspora mitochondrial domain. The reaction products of both the natural and the synthetic lariat's self-cleavage reactions are the same (i.e. 2',3'-cyclic phosphate and a free 5'-hydroxyl termini) but our lariat-RNAs self-cleave three order of magnitude slower than that of the natural counterpart.
Thus the successful design and synthesis of the lariat-RNAs and their analysis by high-field NMR spectroscopy, energy minimizations and molecular dynamics study in water led us to show for the first time the geometrical requirements and features of the self-cleaving site of the self-cleaving strand in the catalytic RNA cleavage reaction. Clearly, the importance of this work lies in the synthesis of artificially designed RNA catalyst as a gene-directed drug.
We for the first time introduced the 2',3'-double bond chemistry in the sugar moiety of a nucleoside as a handle for further functionalization. This has led us to show that various electron-withdrawing groups (CN, PhSO2, PhSeO2, NO2) conjugated to the 2',3'-double bond can be uniquely used to derivatize 2'- and/or 3'- carbons of nucleosides [103, 156, 165, 171, 175, 179, 181, 184, 192, 232] for structure-activity studies as potential antiviral or antitumor agents.
We subsequently introduced intramolecular free-radical chemistry in nucleoside chemistry to produce various 2',3'-sugar-fused heterocycles, which produced various 2'- or 3'-C-branched nucleosides [182, 194, 200, 210, 211, 215, 216, 232, 233, 235, 236].
We also introduced the cyclodipolar addition reactions (to methylnitrones and 3'- nitro-2',3'-vinyl nucleosides) in nucleoside chemistry for the first time [211, 221, 222] to produce various 2'- or 3'-spiro-nucleosides and sugar ring-fused nucleosides, respectively.
We carried out the first diastereospecific synthesis of [3.3.0]- and [3.4.0]-a-cis- fused-carbocyclic nucleosides directly by unsymmetrical modifications of the 2'- and 3'- hydroxyls in seco-nucleosides .
We devised a one-step synthesis of this complex nucleoside with an yield of 76% . This opened door for exploration of its rich synthetic, physical organic chemistry and spectroscopic as well as the kinetic properties of this chemically interesting hypermodified nucleoside [146, 147, 149, 155, 168, 254].
Finally, it emerged from this study that the Y-nucleoside is a derivative of the thermodynamically unfavored N3-H tautomer. The biological consequence of the thermodynamically unstable natural Y- nucleoside is the extreme lability of its glycosyl bond under very mild acidic conditions, which constitutes a switch for the deactivation of the codon function of tRNAphe. This new insight in the inherent thermodynamic unstability of natural Y-nucleoside suggested that guanosine is most probably its biosynthetic precursor.
We have recently developed the first example of an universal aromatic cyclic five- membered P(III) phosphitylating agent, 5-chloro-benzoxazaphosphole [247, see 270 for a review of the field], which allows stepwise activation of various functions of the phosphitylating agent to introduce three different nucleophiles in tandem to give various thio analogs of biologically-occurring assymmetric biophosphates such as Sp and Rp isomers of P1,P4-(diribonucleoside 5')-1-thiotetraphosphate (ApspppA), Sp and Rp isomers of P1,P3-(diribonucleoside 5')-1-thiotriphosphate [ApsppA], Sp and Rp isomers of 5'-phosphoryl-adeninyl-(2''5')-thiophosphoryl)adenosine [ppp5'A2'ps5'A], Sp and Rp isomers of P1-(N7-methylguanosine 5')-P3-(adenosine 5'-)-1-thiotriphosphate [m7GpsppA], ], Sp and Rp isomers of 5'-diphosphoryl adenosine-2'-(thiophosphoryl)- 5'-(N7-methylguanosine) [pp5'A2'ps5'm7G] and 5'-diphosphoryl adenosine-3'- (thiophosphoryl)-5'-(N7-methylguanosine) [pp5'A3'ps5'm7G].
We have achieved a higher stability of the DNA duplex by linking the oligonucleotide to polyaromatic groups [212, 219, 242, 269, 271] or by tethering the oligonucleotide with spermine or spermidine group  or by changing the nature internucleoside bridge to aliphatic linkers . It has been found that the underlying mechanism for the enhanced stabilization of their DNA duplex is owing to the fact that tethered polyaromatic system squeezes water out from the minor and major groove of the DNA duplex [212,219,242], thereby stabilising the base-paired hydrogen bonds.
Another way to increase the stability of an AON/RNA duplex is to incorporate modified nucleotides into the AON strand with a modification that makes the B-type DNA more similar to an A-type RNA. This can be achieved by incorporating nucleosides with a sugar modification that locks the sugar in N-type conformation present in RNA. We achieved this goal with 1',2'-oxetane [313, 315, 329, 333, 334, 336, 340, 350, 351, 357, 360], 1',2'-azetidine [363, 369, 371], 2'-N,4'-C-ethylene- (azaENA) [366, 369, 371, 373, 374, 384] and five and six-membered 2',4'-carbocyclic [372, 374, 377, 384] modification of the ribofuranosyl ring. The 1',2'-oxetane modification pushed the sugar into North-East type conformation (40º < P < 43º, 35º < m < 37º) and met the following antisense criteria: (i) optimal target binding (ii) efficient RNase H recruitment (mixmer strategies) (iii) 3'-exonuclease resistance (iv) blood serum stability (v) endonuclease resistance (Oxetane-T and -C only) upon combination of oxetane-modification and 3'-conjugation of dipyridophenazine (DPPZ). The 1',2'-azetidine locked the sugar North-East type (44º < P < 54º, 29º < m < 33º) conformation and (i) improved RNA target affinity (Tm increase of 1 to 2 °C per modification compared to isosequential oxetane modified counterpart), resulted in: (ii) higher RNase H catalytic activity compared to that of the native AON/RNA duplexes (iii) higher 3'-exonuclease stability and (iv) higher blood serum stability (degraded in 12 h compared to the unmodified AON which is fully degraded in 4 h). The incorporation of azaENA constrained nucleosides locked the furanose ring in North-type conformation (7º < P < 27º, 44º < m < 52º) and fulfilled three important antisense criteria: (i) improved RNA target affinity (Tm increase of 2.5 to 4 °C per modification), (ii) RNase H cleavage rate comparable to that of the native AON/RNA duplexes (iii) higher blood serum stability (~85% stable over 48 h compared to the unmodified AON which is fully degraded in 4 h). The 2',4'-carbocyclic modification leads to further increased nuclease stability in blood serum (stable >48h) . In continuation of these studies, a hyper-contrained methylene bridged hexopyranosyl nucleoside (BHNA) was also synthesized  and incorporated into AON . It was found that this modification decreased the thermal stability of the AON/RNA duplex but resulted in increased blood serum stability.
It is possible to extract 3JHH and nOe volumes for only up to 12mer duplex DNA in an unambiguous manner, but it is simply impossible to collect all of these information in a non-prejudicial manner from a larger DNA molecule. These problems have been found to be associated with spectral overlap which becomes more and more complex due to overcrowding of resonances, particularly from the repeating pentose moieties, with increasing chain length.
We are the first to tackle this problem [94, 98, 125, 133, 136, 203, 207, 224, 235, 251, 256, 257, 261, 262] by substituting proton (1H) with deuteron (2H) chemically in a chosen domain (i.e. non-uniform isotope labelling) and extracting the necessary information arising from the shorter NMR-visible non-deuteriated part (1H-NMR window) (Fig. 1). By incremental shift of the 1H-NMR window in a series of DNA or RNA oligomers with identical sequence (Fig. 1), he has shown that it is possible to put together the total structural information of a much larger oligonucleotide (a 20-mer) than what has been possible prior to this work [224, 235]. The most important part in this concept is that two 1H-NMR windows in two oligomers of the series (Fig. 1) should have at least an overlapping nucleotide residue with specific chemical shifts in order to be able to correlate protons from both windows with respect to the same nucleotide reference point (i.e. same proton resonances in both NMR-windows). Our approach of specific stereospecific deuteriation of sugar residues and their specific incorporation into the selected parts of oligo-DNA should allow the probing of the geometry of the short stretch of the core part in a larger DNA duplex (e.g. a gene or the site of interactions between a promoter site of a specific gene and RNA-polymerase) which would help us to understand how the structure and dynamics of a specific core part of a DNA duplex changes as the DNA duplex chain becomes longer!
We have also recently successfuly shown the application of the Uppsala "NMR- window" concept by solving the solution NMR structure of a 21-mer RNA hairpin [256, 261] that constitutes the recognition signal for the RNA polymerase binding. This Uppsala "NMR-window" concept has been also now employed to solve the NMR structure of a 31-mer RNA subunit of the catalytic RNase P RNA , which has uniquely shown its recognition elements and the dynamics of the active site. Work is now in progress to solve NMR structures of large biological functional DNA and RNA molecules in order to understand the molecular basis of the biological function.
The solution conformation of these duplexes (1) - (4) by 500 MHz 1H-NMR, iterative hybrid relaxation matrix method combined with NOESY distances and torsion angle restrained molecular dynamics, have shown that the Pzn residue stacks with both residues of the neighbouring G-C base-pair in the duplex (2) and contributes to its strong stabilisation, while the Pzn residue in (4) do not stack with the neighbouring G-A mismatch base-pair and adopts at least three different conformations in the NMR time scale. We have addressed for the first time using a combination of NOESY and ROESY experiments [219, 220], how do the stabilitities of duplexes (1) - (4) depend upon (a) the water activity in and around DNA, (b) the strength of hydrogen bond by Watson-Crick base-pairing, and (c) the stacking behaviour. We provided the first unequivocal evidence that the stability of DNA duplex does not necessarily increase with a higher abundance of water inside the minor or the major groove in contrary to the common belief.
A comparison of the relative NOE intensities of both the aromatic and methyl protons with water in the set of four duplexes (1)-(4) suggest that it is only the least stable mismatched duplex (3) (Tm = 25 °C) which has a continuous spine of hydration and ribbon structures through the core part and the terminals, which have been earlier implicated in stabilising the B-form of DNA. Our NMR studies [219, 220] have led us to conclude for the first time that as the stability (Tm) of DNA duplex increases, the exchange-rate of the imino-protons decreases considerably, the life-time increases and the water activity in the minor groove decreases. These data for the first time showed that the intrinsic stabilisation of DNA duplex depends both upon the economy of water activity, which dictate the strength of base-pairs, and the intramolecular base stacking interactions.
Subsequently, the structure of a Pzn promoted stabilised DNA-RNA hybrid duplex (these structures are formed by the viral reverse transcriptase promoted transcription of viral RNA) has also been worked out .
A recent study of the exchange rate of the imino protons in a DNA duplex at different pH and temperature from our lab showed for the first time  that the relative exchange of imino protons of the base-pairs in DNA duplex is more rapid when there is an abundance of water at the first spine of hydration. This work also showed that the core of the DNA duplex is by and large devoid of water, and the energy penalty of water entering the core is very high, thereby providing the first evidence that the water poisoning as the principal factor for base-pair mismatch, frame-shift and mutation in our DNA replication machinery.
A study of DHš and DSš contribution to the free-energy of pseudorotational equilibrium in pentofuranoses 5 - 7 and pentofuranose moieties in nucleosides 10 - 18 allowed us to dissect [213,217] the contributions of various stereoelectronic effects (gauche versus anomeric effects) of exocyclic substituents on pentofuranose moiety.
We have shown that the gauche effect of O4'-C4'-C3'-O3' fragment drives the pseudorotational equilibrium to the S type(2'-endo-3'-exo) conformations while the gauche effect of O4'-C1'-C2'-O2' fragment pushes the pseudorotational equilibrium to the N (3'-endo-2'-exo). He showed that these gauche effects are the strongest factors responsible for the drive of N S pseudorotational equilibrium. The strength of the gauche effect of O4'-C4'-C3'-O3' fragment in abasic sugars 5 - 7 is further tuned by the presence of a heterocyclic base at C1' in nucleosides 10 - 18. Relatively weaker anomeric effect of the heterocyclic base drives the N S equilibrium to the N-type sugar.
The assessment of the relative strengths of the anomeric effects in 2',3'-dideoxy, 2'-deoxy- and ribo-b-D-nucleosides has also shown that the anomeric effect of the cytosine base is stronger than the anomeric effect of adenine or guanine base. Our NMR derived experimental data have shown for the first time that the anomeric effect is considerably reduced as O4' experiences the electron-withdrawing effect(s) of 2'(3')- hydroxyls [238, 239]. The differences in the conformational preferences found in purine and pyrimidine ribonucleosides were additionally attributed to the distinct relative strength of the gauche effect of N-C1'-C2'-O2' fragment. The preference for the gauche orientation of N-C1'-C2'-O2' fragment and therefore S type sugar conformation is affected by the nature of purine or pyrimidine glycosyl nitrogen atom.
These detailed studies for the first time were able to provide the following quantitative experimental DHš and DSš [213, 217, 228, 230, 231, 238, 239] for different stereoelectronic effects due to anomeric and gauche effects which are of considerable importance to understand the oligosaccharide and oligonucleotide structures and also in the construction of the force-field for the energy minimization using molecular mechanics and molecular dynamics: The effect of [5'CH2OH] (+1.5 kJ/mol ±0.8), gauche effect of [O4'-C4'-C3'-O3'] (-6.8 kJ/mol ±0.7), anomeric effect of adenine (+1.7 kJ/mol ±1.0), anomeric effect of guanine (+2.9 kJ/mol ±1.0), anomeric effect of cytosine base (+5.0 kJ/mol ±1.0), gauche effect of [O4'-C1'-C2'-O2'] (+5.7 kJ/mol ±1.3), gauche effect of [N(purine)-C1'-C2'-O2'] (-7.7 kJ/mol ±1.5) and gauche effect of [N(pyrimidine)-C1'- C2'-O2'] (-2.9 kJ/mol ±1.8) on DHš of pseudorotational equilibrium in abasic sugars 5 - 7, 2',3'-ddN 10 - 13, 2'-dN 14 - 17 and riboN 18 - 21.
Basing on the above work, we went on to show  that distinct conformational preferences observed for the pentofuranosyl moieties in various 3'-substituted-2',3'- dideoxythymidine derivatives are closely related to the strength of the 3'-gauche effect, which is directly dictated by the electronegativity of the 3'-substituent. It is our work that allowed to experimentally determine the efficiency of the 3'-gauche effect in a quantitative manner with the help of simple linear calibration curves that correlate the gauche effect enthalpy and the group electronegativity (c) of the 3'-substituent.
We were able to show for the first time how does the 3'-phosphate drive the sugar conformation in DNA ? It emerged from our work  that while the 3JC4'P, 3JC2'P, 3JH3'P, 3JCH3P and 3JCH2P coupling constants in simple model systems 26 - 29 remain unchanged over the whole temperature range of 278 - 358 K, considerable change of endocyclic 3JHH coupling constants have been found to take place, which, for the first time, unequivocally show that the change of the North (3'-endo-2'-exo) South (3'-exo-2'-endo) sugar pseudorotamer equilibrium is independent of the change of the phosphate backbone torsion.
On the other hand, we showed that in RNA, the unique interaction of 2'-OH with the vicinal phosphate act as a molecular switch between two sugar-phosphate state equilibrium: N,et S,e-. This 2'-OH interaction stabilises S-type sugar and e- conformers (the "ON-Off " switch) in a cooperative manner over N-type sugar and et conformers .
Thus the free-energy of the protonation deprotonation or metallation demetallation equilibrium affects the electronic character of the nucleobase as a result of a change of the environment, which alters the stereoelectronuc gauche and anomeric effects, and concomitantly changes the sugar pseudorotational equilibrium in both DNA and RNA [252, 263, 272]. But this change of the sugar conformation is further transmitted to drive the phosphate backbone conformation only in RNA [252, 278, 301, 310].
We have recently shown that C-nucleosides have also intrinsic anomeric effect just as in N-nucleosides [266, 267, 273], and indeed the free-energy of this anomeric effect is transmitted to drive the constiuent sugar conformation as the protonation deprotonation equlibrium of the C-aglycone changes as a function of the pH of the medium.
(A) Determination of pKa of 2'-OH in Ribonucleosides and Ribonucleotides The 2'-hydroxyl group has major structural implication in that it is involved in recognition and catalytic properties of RNA. In order to understand the specific reactivity of certain phosphate function towards stereospecific transesterification reaction, it is important to know the pKa of 2'-OH. We have determined the experimental pKa of 2'-OH in 18 different nucleosides and nucleotides by pH-dependent 1H-NMR analyses , and found that different substitution of the pentofuranose ring would make general acid-base character of 2'-OH different, which in turn may dictate its differential participation in site-specific splicing reaction. Recently, we have measured  the pKa values for internucleotidic 2'-OH of eight different diribonucleoside (3' -> 5') monophosphates by 1H-NMR. By comparing the pKas of the respective 2'-OH of monomeric nucleoside 3'-ethyl phosphates with that of internucleotidic 2'-OH in corresponding diribonucleoside (3' -> 5') monophosphates, it has been confirmed that the aglycons have no significant effect on the pKa values of their 2'-OH under our measurement condition, except for the internucleotidic 2'-OH of 9-adeninyl nucleotide at the 5'-end (ApA and ApG), which is more acidic by 0.3-0.4 pKa units. Since interaction of 2'-OH group with the solvent has major structural implications in the recognition, processing, and catalytic properties of RNA, intra- and intermolecular H-bonding of 2'-OH in adenosine 3'-ethylphosphate, 3'-deoxyadenosine, and adenosine as well as the nature of hydration and exchange processes of 2'-OH with water have also been investigated .
(B)Studies on the non-covalent interactions (stereoelectronics, electrostatics, stacking) in the self-assembly of DNA and RNA The overall stability of nucleic acids, as well as their complexes with a ligand, is considered as a sum of contributions of intermolecular electrostatic interactions, hydrogen bonding, van der Waals and hydrophobic interactions. We have recently given concrete experimental evidences through pKa (as measurable entity) measurements [338, 339, 341, 344, 349, 355, 361, 364] that the electrostatic cross-talk between neighboring aglycones plays an important role in steering both intra- and intermolecular forces culminating into the self-assembly of both single-stranded and homo and hetero duplexes of DNA and RNA. We indeed could show that because of cross-modulation of the aromatic character (evidenced by a change of pKa) amongst the nearest-neighbors as a result of electrostatic cross-talk, a clear pKa modulation takes place [339, 341, 344]. The generality of this pKa modulation was also clearly shown in a much simpler coupled system consisting of pyridine-benzene, covalently linked by linker, which was transformed to pyridinium-benzene by simple change of the pH. The change of the aromatic character from pyridine to pyridinium was electrostatically registered by the phenyl group in the steric proximity by showing the pKa modulation . Through these investigations we have shown that the shift in pKa values is indeed an important source of information about neighboring charges, electrostatics, structural perturbation as well as partial charge distribution over the whole molecule, and differential hydration of the microenvironment.