Studies on nucleic acids chemistry

Abstract: This thesis is divided into three parts: Part 1: Synthesis and conformational analysis of cyclic RNAs, and comparison with lariat RNAs modelling the lariat formed during processing of Group II and Nuclear pre-mRNA. Synthesis of a hexameric lariat RNA model with a tetranucleotidyl "2' → 5' loop" and a dinucleotidyl "3'-tail" has been accomplished. The hexameric lariat and its heptameric analogue displayed unique self-cleavage giving products, mimicking those obtained in the self-cleavage reactions of catalytic RNA (ribozyme). To elucidate the role of the "3'-tail" in the observed self-cleavage, two cyclic tetrameric (2' → 5' at A1) and (3' → 5' at A1) RNAs were synthesized. A comparison of the conformations adopted by them and the hexameric lariat-RNA highlighted that just an ethyl phosphate as the "3'-tail" is adequate to engineer the local geometrical changes that are required for the self-cleavage reaction. Part 2: Synthesis and utility of cyclic phosphitylating agents in the one-pot synthesis of biologically relevant phosphorothioates. The synthesis of new aromatic cyclic five-membered P(III) phosphoramidite is reported. MAC activation of these phosphitylating reagents made it feasible to generate unsymmetrical trisubstituted phosphates by stepwise reacting the activated phosphole with three different nucleophiles. The use of the 5-chloro phosphole analog was demonstrated in the one-pot synthesis of various biological oligophosphates such as P1,Pn-(diadenosine 5')-polyphosphates, P1-(N-7-methylguanosine 5')-P3-(adenosine 5')-1-thiotriphosphate, nucleoside 5'-polyphosphates, nucleoside 2',3'-cyclic phosphorothioate, nucleoside phosphorothioates and trinucleoside monophosphate. Part 3: Synthesis and physicochemical studies on duplexes and triplexes of DNA-conjugates: A class of potential antisense and antigene agents. DNA-conjugates have found utility in antigene and antisense strategies as repressors of gene expression at the transcriptional and translational levels. The first synthesis of the C-branched spermine is herein reported. It was covalently linked through a phosphate linkage ontothe 5'-end of a ssDNA, and through the 2'-phosphate of the arabinose-Uracil to the medial and the 3'-end of ssDNA. The resulting DNA conjugates augmented the thermal stability of their duplexes and triplexes formed with ssDNA and dsDNA. Additionally, thirteen different planar hydroxy alkylated polyarenes with different geometries, bulk and π-electron densities were synthesized. Tethering them through a phosphate linkage to the 5'-terminus of ssDNA enabled us to demonstrate the influence of varying the electronic and bulk characteristics of these polyarenes on the thermal stability and fluorescence properties of the resulting hetero/homoduplexes and the triplexes. CD displayed no gross changes in the native hetero/homoduplex global structure on tethering these fluorophores. It has also emerged that these 5'-tethered fluorophores assist in pre-organization of DNA-conjugates owing to which they adopt a less random coil structure than their native counterparts. All heteroduplexes tested underwent RNA excision by RNase H. Finally, a significant hurdle in the antisense strategy, which is that of the kinetic inaccessibility of mRNA, is addressed using a small 20-mer RNA. This 20-mer RNA adopts a duplex structure which did not form a complex with our DNA-conjugates, while its DNA counterpart which formed a hairpin did.