DNA with zwitterionic and negatively charged phosphate modifications: formation of DNA triplexes, duplexes and cell permeability studies

Two phosphate modifications were introduced into the DNA backbone using Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and the sulfonyl azides, 4-(azidosulfonyl)- N,N,N -trimethylbutan-1-aminium iodide (N+ azide) or p -toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts- modification in the DNA sequence. Incorporation of these N+ and Ts- modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts- modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs where the modification is in the middle of the sequence). Incorporation of N+ modifications led to the formation of duplexes whose thermal stability was less dependent on ionic strength than native DNA duplexes. Thermodynamic analysis of melting curves revealed that it is a reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell permeability studies showed that Ts- ONs diffuse into the nucleus of mouse fibroblast NIH3T3 cells without the need for transfection reagents. In contrast, N+ ONs were concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts- modified ONs are promising for various in vivo applications.