(DNA or RNA) and is thus an intrinsic property of tc-DNA/DNA- versus tc-DNA/RNA-backbone junctions. Thermodynamic data. We calculated thermodynamic data of duplex formation for oligonucleotides 18 and 20, con- taining discontinuous or continuous tc-modiﬁcations, re- spectively, with DNA or RNA as complements and compared them with the duplexes containing the unmodi- ﬁed RNA strand 12 or the fully tc-DNA-modiﬁed strand 11 (Figure 4 and Supplementary Table S2). With DNA as complement, the enthalpy of duplex formation is least fa- vorable in the case of discontinuous tc-modiﬁcation, recovers to some extent in the case of continuous tc-modiﬁcations and is most favorable in the case of the fully modiﬁed tc-DNA/DNA duplex. As expected this is to some extent counterbalanced by entropy compensation (26,27), leading to G values (25 C) that are in line with the measured Tm-values. A similar tendency occurs with RNA as the complement, however, the relative differences in the enthalpy and entropy terms are smaller as compared with the DNA complement series. This points to the fact that tc-DNA/RNA junctions are associated primarily with a loss of pairing enthalpy H which contributes unfavor- ably to duplex formation. CD spectra of tc-modiﬁed RNA duplexes. While single in- corporations of tc-residues into the RNA backbone change the CD spectra with complementary DNA and RNA only marginally, there exist substantial differences between duplexes containing tc-DNA residues in a discon- tinuous (18) or continuous (20) fashion (Figure 5). Both show reduced positive ellipticities around 270 nm compared with the unmodiﬁed duplexes. While the struc- ture of duplexes with RNA are more similar and A-like, irrespective of the distribution pattern of the tc-residues, the analogous duplexes with DNA as complement are structurally more diverse showing a distinctly different pattern in the low wavelength region between 210 and 240 nm, and a gradual change of the overall duplex struc- ture from a mixed A,B-like conformation to a more A-like conformation with increasing number of tc-residues. This shows that the number of tc-DNA/RNA junctions Nucleic Acids Research, 2011, Vol. 39, No. 1 377 in the backbone leads to signiﬁcant changes in duplex conformation, especially with DNA as a complement. Modeling of tc-DNA and -RNA junctions. To correlate the thermal and thermodynamic properties with structure on a more detailed level, we performed molecular dynamics calculations of duplexes of the mono-modiﬁed oligo- nucleotides 3 and 14 with complementary DNA and RNA (Figure 6). It turned out that a tc-T residue in the DNA strand 3, paired to DNA (Figure 6a), does not signiﬁcantly alter the overall B-DNA conformation expected for such a duplex. No unusual backbone torsion angles or nucleoside con- formation are observed for the two ﬂanking natural nu- cleotides in the strand. The tc-T residue shows an interchange in the conformation of torsion angle and to gauche, trans instead of the usual trans, gauche. This interchange is structurally compensatory in nature and very closely reproduces the local conformation observed in an X-ray structure of a tc-DNA modiﬁed DNA duplex (28). Thus, there seems to be no conﬂicting situation at the DNA/tc-DNA junction, which is well re- ﬂected by the mostly invariant Tm/modiﬁcation of duplexes containing single incorporations in a discontinu- ous way. A similar situation is encountered when 3 is paired to complementary RNA (Figure 6b). Also in this case, the ﬂanking natural deoxynucleotides are in their normal 20-endo conformation and for the tc-T residue the same changes apply as before. The complementary RNA strand shows all nucleotides in the 30-endo conform- ation, giving rise to a mixed duplex conformation. Also here, no structural violations within and around the modi- ﬁcation occurs which is again reﬂected in the Tm/ modiﬁcation data (Table 1), which are positive for all duplexes containing single or multiple tc-DNA/DNA junctions. If tc-nucleosides are incorporated into RNA, as in 14, the picture changes signiﬁcantly. In the duplex of 14 with complementary DNA (Figure 6c), the rC-nucleotide 30 to the tc-T modiﬁcation occurs in the unusual 20-endo conformation with an unusual torsion angle and phosphodiester conformation. The 50-rG-residue occurs more or less in a canonical A-RNA Figure 4. Graphical representation of the thermodynamic data of duplex formation (standard conditions, 25 C, kcal/mol) from van’t Hoff analysis of the UV-melting curves of oligonucleotides 12, 18, 20 and 11 in duplex with either complementary DNA (a) or RNA (b).