376 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 2. 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 1, 7, 9 and 11 in duplex with either complementary DNA (a) or RNA (b). Figure 3. CD spectra of oligonucleotides 1, 3, 7 and 9 in complex with complementary DNA (a) and RNA (b). Experimental conditions as for UV-melting curves, T = 25 C. investigated their thermal melting properties in duplex with complementary DNA and RNA (Table 2). When introducing a single modiﬁcation into the center of RNA strand, as in 13 and 14, a signiﬁcant drop in Tm between 3.7 and 5.3 C is observed irrespective of whether the complement is RNA or DNA. The 50- or 30-end modiﬁcations are slightly stabilizing with DNA as a complement. With complementary RNA, only the 30-end modiﬁcation slightly stabilizes the duplex. Again, discontinuous tc-units destabilize duplex formation with complementary DNA and RNA by 3.0 to 5.1 C/mod. On a per-residue level, the destabilization is more pronounced with a lower number of modiﬁcations. Continuously increasing the number of consecutive tc-units from two to eight is associated with changes in Tm/modiﬁcation from 2.1 to +1.7 C with both, the DNA and RNA complement. Thus, there are considerable differences of tc-modiﬁed RNA as compared with tc-modiﬁed DNA in thermal stabilities, speciﬁcally with RNA as a complement. While modiﬁcation of a DNA backbone with tc-residues stabilizes in all cases a duplex with RNA, irrespective of the number of tc-DNA/DNA junctions in the backbone, the same modiﬁcation in the RNA backbone is distinctly destabilizing with increasing number of tc-DNA/RNA junctions in the oligonucleotide. In other words, Table 2. Cooperative and anti-cooperative effects on thermal duplex stability with DNA and RNA complements of oligoribonucleotides containing an increasing number of tc-DNA residues 12 13 14 15 16 17 18 19 20 21 11 Sequencea r(AACUGUCACG) r(AACUGUcACG) r(AACUGtCACG) r(p-aACUGUCAC) r(AACUGUCACg) r(AACtGUcACG) r(AaCtGtCaCG) r(AACUGtcACG) r(AActgtcaCG) r(AactgtcacG) tc(p-aactgtcacg) Tm ( C, 260 nm) with DNA complementb,c Tm ( C, 260 nm) with RNA complementb,c 41.3 37.6 ( 3.7) 36.0 ( 5.3) 41.9 (+0.6) 42.9 (+1.6) 31.0 ( 5.1) 29.3 ( 3.0) 37.0 ( 2.1) 45.7 (+0.7) 54.8 (+1.7) 58.5 (+1.7) 54.0 49.9 ( 4.1) 49.0 ( 5.0) 53.3 ( 0.7) 55.0 (+1.0) 45.2 ( 4.4) 41.3 ( 3.2) 49.6 ( 2.2) 54.9 (+0.1) 63.1 (+1.1) 64.3 (+1.0) aCapital letters: ribonucleotides; small letters bold: tricyclo-nucleotides; 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. discontinuous introduction of tc-residues destabilize duplexes, whereas a continuous substitution pattern in- creases duplex stability. Again, this does only marginally depend on the nature of the complementary strand