Table 1. Cooperative and anti-cooperative effects on thermal duplex stability with DNA and RNA complements of oligodeoxyribonu- cleotides containing an increasing number of tc-DNA residues 1 2 3 4 5 6 7 8 9 10 11 Sequencea d(AACTGTCACG) d(AACTGTcACG) d(AACTGtCACG) d(p-aACTGTCACG) d(AACTGTCACg) d(AACtGTcACG) d(AaCtGtCaCG) d(AACTGtcACG) d(AActgtcaCG) d(AactgtcacG) tc(p-aactgtcacg) Tm ( C, 260 nm) with DNA complementb,c Tm ( C, 260 nm) with RNA complementb,c 46.0 48.3 (+2.3) 45.6 ( 0.4) 45.6 ( 0.4) 48.2 (+2.2) 48.3 (+1.1) 45.0 ( 0.2) 51.0 (+2.5) 54.3 (+1.4) 56.9 (+1.4) 58.5 (+1.3) 43.6 48.3 (+4.7) 46.0 (+2.4) 44.6 (+1.0) 48.2 (+4.6) 51.0 (+3.7) 49.3 (+1.4) 51.6 (+4.0) 59.9 (+2.7) 63.9 (+2.5) 64.3 (+2.1) aCapital letters: 20-deoxynucleotides; small letters bold: tricyclo-nu- cleotides; p, phosphate. bConditions: c, 2 mM in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0. Estimated error in Tm: ± 0.5 C. cValues in parenthesis: Tm/modiﬁcation. modiﬁcations were introduced at the 50- and 30-ends and in the center of the sequence (2-5) followed by sequences containing either two or four tc-units spaced by natural nucleotides (6, 7) or two, six and eight tc-residues in a continuous fashion (8-10). The unmodiﬁed (1) and the fully modiﬁed (11) oligonucleotides served as references. Tm-data. With DNA as a complement, single modiﬁca- tions in the center of the sequence (2, 3) revealed devi- ations in Tm ranging from 0.4 to +2.3 C relative to the unmodiﬁed duplex. The differences are likely due to base-speciﬁc effects. The situation with RNA as a comple- ment is similar with a distinctly higher tendency to duplex stabilization. The 50-end modiﬁcation leads to no major change in Tm with DNA as complement, while 30-end modiﬁcation stabilizes both duplexes with DNA or RNA complements by +2.2 and +4.6 C, respectively. The Tm-data of the 50-end-modiﬁed duplexes have to be interpreted and compared carefully, as for chemical sta- bility reasons they contain a 50-phosphate group which is not the case for the reference duplex containing 1. Increasing the number of tc-residues from two to four in a discontinuous way (at least one natural nucleotide in between) has a Tm-neutral to slightly stabilizing effect with DNA as complement ( 0.25 to +1.1 C/modiﬁcation) and deﬁnitely a stabilizing effect with RNA as the complement (+1.4 to +3.7 C/modiﬁcation). More tc-modiﬁcations, as in 7, are clearly less stabilizing on a per-residue basis as compared with fewer modiﬁcations in a similar arrangement as in 6. Extending the number of continuous tc-residues from 2 to 10 (8–11) is associated with increasing duplex stability with both DNA and RNA as the complement. The stabilization on a per-residue basis ranges from+2.5 to+1.4 C with DNA as complement and from+4.0 to+2.5 C with RNA as complement and clearly levels off with increasing number of modiﬁcations. Thermodynamic data of duplex formation. From van’t Hoff analysis of representative melting curves of Nucleic Acids Research, 2011, Vol. 39, No. 1 375 oligonucleotides 1, 7, 9 and 11 in complex with comple- mentary DNA or RNA, we calculated the transition enthalpies ( H, G) as well as the entropy changes (TDS) under standard conditions (25 C) (Figure 2) (23,24). Detailed numerical values are available in Supplementary Table S2. In all cases the free enthalpies of duplex formation ( G) reﬂect the order of thermal stabilities as represented by the Tm-values. With DNA as complement, oligonucleotide 7 containing four spaced tc-DNA residues leads to a drop in both the H and TDS term compared with 9 containing six continuous tc-residues. The most favorable pairing enthalpy H was found for the fully modiﬁed oligonucleotide 11. With RNA as a complement a similar trend is observed, however, there is much less variability in the enthalpy and entropy terms. Thus, an increasing number of tc-DNA/ DNA junctions in the backbone of the modiﬁed strand is associated primarily with a loss of pairing enthalpy H which is compensated to some extent by the entropy term. This is especially signiﬁcant for duplexes with DNA as complement. CD spectra of modiﬁed duplexes. We determined CD spectra of selected modiﬁed oligonucleotides with either complementary DNA or RNA (Figure 3). With DNA as complement, and compared with the unmodiﬁed duplex, already the introduction of a single tc-T unit, as in 1, leads to an increase of the positive cotton effect at around 270 nm and a decrease of the negative cotton effect at 245 nm. The duplex containing tc-units in a discontinu- ous way reinforces this behavior. The duplex containing six continuous tc-units shows a slight red shift of the positive cotton effect and an overall shape with increased A-conformation character. Thus, increasing the number of tc-units is associated with a gradual transition from a B-type to an A-type duplex and this behavior is more ex- pressed for duplexes containing continuous tc-units. This is in agreement with the structural features of a DNA duplex containing a fully modiﬁed tc-DNA strand (25). With RNA as a complement there is almost no structural difference between the unmodiﬁed DNA/RNA duplex and that containing various tc-units in different positions. From the decrease of the intensity of the negative band at 240 nm with increasing number of tc-units in the DNA strand it can be anticipated that there is an additional shift from a mixed, A–B-conformation to more A-character. This is in full agreement with earlier observations from CD spectroscopy and molecular modeling which describe tc-DNA structurally as an RNA and not a DNA analog (25). Interestingly, no differences between duplexes containing 7 and 9 are observed, indicating non-detectable structural differences between discon- tinuous or continuous tc-modiﬁcation of a DNA strand in complex with RNA. Tc-DNA modiﬁcations in the RNA backbone Tm-measurements. To complete these structural investiga- tions, we prepared the analogous series of modiﬁed RNA oligonucleotides 12–21 with tc-DNA modiﬁcations and