Physico-chemical properties of 5′-polyarene tethered DNA-conjugates, and their duplexes with complementary RNA

Fluorophores 1-13 when covalently linked to the 5'-terminus of 9-mer ssDNA (as in ssDNA-conjugates 16-28) enhance the stablity of the duplexes with both RNA (29) and DNA (30) targets compared to the natural counterparts, which, for the first time, demonstrated the effect of the bulk and the x-electron density of various 5'-tethered fluorophores on the heteroduplex stability. It has been found that decreasing the pi-electron density of the fluorophore induces a more favourable pi-pi interaction with the adjacent nucleobase, leading to higher duplex stability. Increasing the surface-area of 5'-stacked fluorophore only increases the thermal stability of the duplex, if it leads to an increase in the area of pi-overlap with the adjacent nucleobase. The fluorescence characteristics show that the tethered-fluorophores stack to the exterior of the terminal nucleobase pair, except for 5'-tethered-alpha-Napthalene (10), which shows dramatic enhancement in fluorescence as normally observed for the minor groove binders. CD data shows that tethering the DNA with different fluorophores at the 5'-end did not make any gross changes in the helical structure of the duplexes of DNA-conjugates with RNA and DNA targets compared to the natural counterparts. It has emerged that the 5'-tethered fluorophores assist in pre-organising the ssDNA-conjugates into a helical conformation more effectively by a stronger fluorophore-nucleobase stacking than in the native ssDNA. All DNA-conjugates tested assisted in cleavage of the complementary RNA strand by RNase H. The RNase H activation by 5'-conjugated DNA-RNA duplex decreased with the decrease of their thermal stabilities, and as they deviated from the structure of the corresponding native DNA-RNA duplex. It has been also found that both native and conjugated DNA-DNA duplexes can indeed block the RNase H activity, thereby reducing the effect of a potential antisense agent. This implies that the palindromic as well hairpin-forming sequences of antisense DNA should be avoided.