A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues Tm and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in Tm when paired to complementary DNA and leads to substantial increases in Tm when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.

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Abstract:We present the synthesis of the iso-bicyclo DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo-DNA synthesis. Dodecamers containing single iso-bicyclo thymidine incorporations, fully modified A-and T-containing sequences and fully modified oligonucleotides containing all four bases were synthesized and characterized. Iso-bicyclo DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self-pairing. Iso-bicyclo DNA forms preferentially B-DNA-like duplexes with DNA and A-like duplexes with complementary RNA as determined by CD-spectroscopy. Self-paired duplexesshow a yet unknown structure, as judged from CD-spectroscopy. Biochemical tests revealed that isobicyclo DNA is stable in fetal bovine serum and does not elicit RNase H activity.