Synthesis of 5'-polyarene-tethered oligo-DNAs and the thermal stability and spectroscopic properties of their duplexes and triplexes

Article abstract of DOI:10.1016/S0040-4020(97)00631-5

Eleven different planar hydroxy alkylated polyarenes 1-11 with different geometry, bulk and electronic characteristics were synthesised, and used for tethering to the 5'-phosphate of a 9-mer and a 18-mer DNA sequences through solid-phase synthesis. The 5'-polyarene-tethered 9-mers 30-40 were tested for their ability to form stable duplexes with four complementary target DNA-strands 25-28 of different length. The 5'-polyarene-tethered 18-mers 44-54 were tested for their ability to form stable triplexes with a 24-mer duplex target 41+42. The T(m) measurements of duplexes at low salt and pH 7.3 showed that the angular nitro phenanthrene and phenanthrene conjugates 31 and 30 gave the highest duplex stabilisations with targets 25 (ΔT(m) 13.8°C and 11.8°C) and 26 (ΔT(m), 12.3°C and 11.9°C). With the mismatch sequence 28, only 30 and 31 gave a high ΔT(m), of 11.6°C and 10.8°C respectively, while lower ΔT(m), values were observed for other conjugates (ΔT(m), -4.0-5.0°C). The T(m) measurements of triplexes between 43-54 and duplex target 41+42 at low salt and pH 7.3, 6.5 and 6.0 without Mg²⁺ showed that the nitro phenanthrene conjugate 45 gave the best triplex stabilisation (ΔT(m), 4.1-5.4°C). The stabilisation of nitro phenanthrene conjugate 45 compared to phenanthrene conjugate 44 increased more remarkably when Mg²⁺ was present: 45 (ΔT(m), 15°C), 44 (ΔT(m), 10°C). These results imply that the electron density of the chromophore influences the π-π stacking interactions between the chromophore and nucleobases, and thereby influencing the duplex and triplex stability. Fluorescence measurements on single strand to double strand transition indicated that the 5'-tethered polyarenes are stacked only on the neighbouring nucleobases of the opposite strand. In case of 5'-9-N-ethylphenazinium conjugate 36, a comparative NMR and fluorescence measurement has unambiguously shown that the tethered phenazinium ion is indeed intercalated between the nucleotides of the opposite target strand 26. Thermodynamic calculations showed the most stable ΔG°(298K) for 30, 31(+targets 25, 26, 28) and 35, 36(+targets 25, 26) compared to the blank 29 ΔΔG°(298K) ~-10kJ mol^-1). The non-palindromic target 27 was shown by T(m) measurements to form a stable tertiary structure, which was very little affected by addition of any 5'-tethered conjugate, thereby showing the importance of the tertiary structures of an in vivo antisense target and its implication in regard to its bioavailablity to complementary antisense probes.