374 Nucleic Acids Research, 2011, Vol. 39, No. 1 guide strand selection by the use of siRNAs with judi- ciously positioned LNA residues (15). Improved antisense strand selection by RISC has also been attained by using small internally segmented siRNAs (sisiRNAs) (16). Meanwhile, a large variety of siRNAs with various chemical modiﬁcations have been tested with encouraging results (17,18). In recent work, we demonstrated that siRNAs contain- ing tricyclo-DNA (tc-DNA, Figure 1) modiﬁcations in various positions in the sequence can improve RNAi (19). During thermal melting analyses of tc-modiﬁed siRNA duplexes, we became aware of a signiﬁcant vari- ability in Tm, depending on the position of the modiﬁca- tions in the sequence. In order to rationalize these effects, we set out to systematically determine the structural and energetic effects of single and multiple tricyclo-DNA modiﬁcations in a DNA- or RNA backbone on its pairing to complementary DNA and RNA. MATERIALS AND METHODS Oligonucleotide synthesis and puriﬁcation Oligonucleotides were synthesized on a 1.3 mmol scale on a Gene Assembler Plus (Pharmacia), using 5-(ethylthio)- 1H-tetrazole (0.25 M in CH3CN) as the activator and following standard phosphoramidite chemistry in the DMT-off mode. The phosphoramidites of the natural DNA-nucleosides were from Vivotide and the 20O- triisopropylsilyloxymethyl (TOM)-RNA-amidites as well as DNA- or RNA-solid supports from Glen Research. Tc-DNA nucleotides were attached to a Amino SynBase CPG solid support (Link Technologies) in analogy to pub- lished procedures (20). Oligonucleotides 1–11 containing only deoxyribonucleosides were deprotected in 33% aqueous NH3 for 16 h at 55 C. Oligonucleotides 12–21 containing ribonucleosides were base- and phosphate deprotected with 8 M methylamine in EtOH/H2O 1:1 for 12 h at 35 C, followed by TOM deprotection in 1 M tetrabutylammoniumﬂuoride (TBAF) in tetrahydrofuran (THF) for 10 min at 50 C and then 6 h at 35 C. All oligo- nucleotides were puriﬁed by ion-exchange chromatog- raphy on a DNAPac PA200 column (Dionex, 4 250 mm); solvents: A: 25 mM Trizma base in H2O, pH = 8.0; B: 25 mM Trizma base, 1.25 M NaCl in H2O, pH = 8.0. Gradient 0–50% B in 30 min. The purity of all Figure 1. Chemical structure of tricyclo(tc)-DNA. tc-modiﬁed oligonucleotides (HPLC control) was >96%. The constitution of all oligonucleotides was veriﬁed by ESI-mass spectrometry (Supplementary Table S1). UV-melting curves Solutions were prepared in standard saline buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.0) with duplex concentrations of 2 mM in 1:1 strand ratio. Thermal melting experiments were carried out on a Varian Cary 100-Bio UV/VIS photospectrometer (Varian Inc.), equipped with a Peltier element at 260 nm with a heating/cooling rate of 0.5 C/min. Tm values were obtained from the maxima of the ﬁrst derivatives of the melting curves using WinUV software. CD measurements CD spectra were recorded on a Jasco J-715 spectropo- larimeter equipped with a Jasco PFO-350S temperature controller, using the same buffer conditions and oligo- nucleotide concentrations as for UV melting curves. All CD spectra were collected at 25 C between 210 to 320 nm, using a wavelength step of 5 nM. Subsequently, the graphs were smoothed with a noise ﬁlter (convolution width: 7) and analyzed using the J-700 control driver. The reported spectra correspond to the average of at least three scans. Thermodynamic parameter Thermodynamic parameters were determined by ﬁtting a simulated curve to the experimental UV-melting curve. The simulated curves were based on a bimolecular system obeying the two-state model (21) and were obtained using our ﬁtting function ‘alpha’ which was built in analogy to published procedures (22). The ﬁtting process was performed with the program Origin 8 (OriginLab). Molecular modeling Molecular modeling was performed with the Amber force ﬁeld as incorporated in the software package HyperChemTM Release 8.0.4 for Windows (Hypercube, Inc.). Only original Amber parameters were used and no explicit water or counterions were included. A distance- dependent scale factor of " = 1 was used. One to four non-bonded interactions were scaled by 0.5. No cutoffs were applied. DNA/DNA structures were built on the basis of a B-DNA, all structures containing RNA on the basis of an A-DNA. All sequences were ﬁrst minimized until the gradient was <0.1 kcal/mol A˚ . The structures were then heated from 0 to 300 K over 200 ps and submitted to 200 ps of unrestrained molecular dynamics at 300 K. After simulation, the structures were minimized again until the gradient was <0.1 kcal/mol A˚ . RESULTS Tc-modiﬁcation of DNA To determine the effects of tc-modiﬁcations in the context of the DNA backbone, we synthesized a series of decamers with a randomized, generic base sequence (Table 1). Single