378 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 5. CD spectra of oligoribonucleotides 12, 14, 18 and 20 in complex with complementary DNA (a) and RNA (b). Experimental conditions as for UV-melting curves, T = 25 C. backbone conformation. The complementary nucleotides on the DNA complement show less structural variation. The Tm/modiﬁcation data (Table 2) clearly show that an increasing number of tcDNA/RNA backbone junctions have an additive negative effect on the Tm. Modeling thus suggests that this negative effect is the consequence of unfavorable conformational changes of the neighboring ribonucleotides. This is essentially also found for the case of 14 paired to complementary RNA (Figure 6d). Also here, the ﬂanking ribonucleotides switch into the higher energy 20-endo conformations, which is again in agree- ment with the Tm/modiﬁcation data (Table 2) which show a negative tendency that is additive with the number of tc-RNA/RNA junctions. In summary, the structural data suggest that the inﬂuence of a tc-residue within a DNA backbone leads to less conformational changes to its nearest neighbors, while a tc-residue in a RNA backbone signiﬁcantly changes its nearest neighbors into higher energy conformations. This is well reﬂected in the corresponding thermal and thermodynamic data set. DISCUSSION Tc-DNA was introduced as a second-generation conformationally constrained oligonucleotide analog of the bicyclo-DNA molecular platform some 13 years ago. While in the past, most biophysical and biological evalu- ations of tc-DNA were performed on the basis of fully modiﬁed oligonucleotides, this is the ﬁrst systematic study of the thermal, thermodynamic and structural effect of single or multiple incorporations of tc-nucleosides in either the DNA- or the RNA backbone. Given the substantial structural changes in a tc-nucleoside compared with a natural nucleoside, it is not surprising that these inﬂuence nucleic acid afﬁnities in a distinct way. In this study, we did not intend to determine base-speciﬁc effects on structure or stability, but we focused on sugar/backbone-speciﬁc changes. We note that base-speciﬁc effects may be operative in addition, but we believe that they will have lower structural or energetic impact. From earlier work, it is evident that fully modiﬁed tc-oligonucleotides stabilize duplexes with complementary Figure 6. Strereoviews of the central section of the duplexes of (a) 3/DNA, (b) 3/RNA, (c) 14/DNA and (d) 14/RNA, containing the modiﬁed tc-DNA- as well as its 30- and 50-ﬂanking natural base pairs after a total of 400 ps dynamic simulation followed by energy minimiza- tion using the AMBER force ﬁeld as implemented in the software package HyperchemTM. DNA and RNA by 1–3 C per modiﬁcation, compared with DNA. Structural investigations by CD spectroscopy, molecular modeling and, more recently X-ray analysis (28), revealed that major deviations in the backbone struc- ture occur within torsion angles and , which show more a trans, gauche than a gauche, trans arrangement as observed for DNA and RNA. This leads to an overall strand conformation that is nearer to an A-conformation (25). Single or multiple substitutions of