a DNA strand by tc-nucleosides in either a continuous or discontinuous way, as investigated in this work, leads to relatively small variations in Tm/modiﬁcation with DNA as complement (Table 1). Nevertheless, a slightly negative tendency with increasing number of tc-DNA/DNA heterojunctions in the backbone is evident. With RNA as a complement, only stabilization occurs which is again less pronounced if the number of heterojunctions in the backbone is high. The CD spectra (Figure 3) clearly show that the structural changes are higher in the duplexes with complementary DNA as compared with RNA. The higher the amount of tc-units in the strand, the more the duplex conformation shifts towards an A-like structure, comparable to that of a fully modiﬁed tc-DNA/DNA duplex. Interestingly, there is a negligible energetic penalty to this, and molecular modeling (Figure 6a and b) suggests that this shift in con- formation is mainly a direct effect of the modiﬁcation itself and not a consequence of structural changes imposed to nearest neighbor natural nucleosides in the strand. The picture is different if the RNA backbone is modiﬁed with tc-nucleosides. Here, a signiﬁcant thermal destabilization is observed when tc-units are spaced by natural ribonucleosides and thus the number of tc-DNA/RNA heterojunctions in the backbone is high. The destabilization is mainly of enthalpic origin. The opposite effect occurs if tc-units are introduced in a con- secutive manner, where an additive stabilization with increasing number of tc-residues is observed. Structural modeling of a tc-DNA heterojunction reveals that with both, DNA and RNA complements, substantial changes of the nearest neighbor nucleosides occur. The most relevant changes are a shift from 30- to 20-endo conform- ation of the adjacent ribonucleotides and changes of the phosphodiester bonds to higher energy gauche, trans con- formations. From the modeled structures (Figure 6c and d), it seems that at least for the nucleotides 50 to the tc-modiﬁcation the adjustment of their ribose conform- ation is driven by minimizing steric conﬂicts between their 20-OH function and the furanose ring of the tc-unit. This conﬂict comes from the dominant trans, gauche arrangement of torsion angle and , imposed by the tricyclo-scaffold, which is of course absent in natural backbone junctions and is substantially reduced in tc-DNA/DNA junctions (Figure 6a and b) because of the replacement of an OH group by a sterically less-demanding hydrogen atom. This structural rational- ization is reminiscent to that invoked in the case of the completely different nucleotidic pairing system glucopyranosyl-NA. The presence of a 20-OH group in this case substantially decreased duplex formation efﬁ- ciency compared with homo-DNA in which this 20-OH function is replaced by a hydrogen atom (‘too many atoms’) (29,30). It is not uncommon that chemically modiﬁed residues can inﬂuence their nearest neighbors in a cooperative fashion in nucleic acid duplexes and has, for example, been found in the case of LNA. It has been shown by high-resolution NMR experiments that nearest neighbor deoxynucleotides next to a LNA unit show a higher Nucleic Acids Research, 2011, Vol. 39, No. 1 379 population of the 30-endo conformation (31–33). This con- formational steering induced by the LNA nucleotides has been rationalized by a change in electronic density at the rim of the minor groove, causing an alteration of the pseudorotational proﬁle of the 30-ﬂanking nucleotide toward N-type conformation. A direct comparison of LNA- and tc-DNA units as strand modiﬁers highlights substantial differences between these two members of the class of conformationally stabilized analogs. While LNA units drive adjacent deoxynucleotides into an N-type conformation, this seems not to be the case for tc-DNA. While tc-DNA/DNA heterojunctions are associated with a slight loss in duplex formation enthalpies, LNA/DNA heterojunctions are in most cases enthalpy stabilized (34). While tc-DNA residues in an RNA backbone drive ribonucleotides more into an S-type conformation, this seems not to be the case for LNA, and also no energetic penalties arise from LNA/RNA backbone junctions. This can be inferred from a steric block antisense experiment in which tcDNA/20-OMe-RNA mixmers were compared with LNA/20-OMe-RNA mixmers (35). It clearly emerged that fully modiﬁed tc-DNA sequences were more active compared with tc-DNA/20-OMe mixmers, while the cor- responding LNA/20-OMe mixmers did not show any signs of compromised activity. Thus, LNA mixes well with RNA or 20-OMe-RNA, while tc-DNA does not. CONCLUSIONS In this systematic study, we found that there are signiﬁ- cant positional effects on Tm upon incorporation of tc-DNA residues into DNA or RNA homo- or heteroduplexes. In particular, introducing tc-residues into an oligoribonucleotide in a discontinuous fashion sig- niﬁcantly decreases the Tm, while continuous tracts of tc-residues or fully modiﬁed tc-oligonucleotides stabilize duplexes. These ﬁndings are of relevance for the design of tc-DNA containing oligonucleotides for therapeutic purposes. For antisense oligonucleotides acting by a steric block mechanism, where high RNA afﬁnity is required, the use of fully modiﬁed tc-oligonucleotides seems to be the appropriate choice. For applications in RNAi, the positional stabilization/destabilization of tc-residues can be used to design thermodynamic asym- metry into a siRNA duplex. For example, it seems advis- able to substitute a passenger strand of a siRNA duplex at the 30-end in a discontinuous way with tc-nucleotides to thermodynamically destabilize the duplex at the 50-end of the guide strand. Similarly the 50-end of the passenger strand should be modiﬁed in a continuous way to thermo- dynamically stabilize the duplex at the 30-end of the guide strand, thus increasing the thermodynamic asymmetry and directing guide strand selection by RISC. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online