Published online 17 August 2010 Nucleic Acids Research, 2011, Vol. 39, No. 1 373–380 doi:10.1093/nar/gkq733 Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes Damian Ittig, Anna-Barbara Gerber and Christian J. Leumann* Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland Received June 30, 2010; Revised and Accepted August 3, 2010 ABSTRACT A series of oligodeoxyribonucleotides and oligoribo- nucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand signifi- cantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy "H. Arranging the tc-residues in a continuous fashion rescues Tm and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in Tm when paired to comple- mentary DNA and leads to substantial increases in Tm when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conform- ation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influ- ence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA con- taining oligonucleotides for applications in anti- sense or RNAi therapy. INTRODUCTION Chemically modiﬁed oligonucleotides are of great current interest as modulators of gene expression in RNA- targeting therapeutic applications (1–3). Their mode of action basically relies on Watson–Crick pairing to a given target RNA, inducing various and complex bio- logical gene-silencing mechanisms that mainly depend on the nature of the chemical modiﬁcation and the RNA target. Typically, single-stranded oligonucleotides modulate the biological function of an RNA via antisense mechanisms by either simply binding to it (steric block inhibitors), by concomitantly promoting its degradation with the help of endogenous enzymes such as RNaseH or by interfering with the splice machinery. Double- stranded oligoribonucleotides on the other hand induce silencing of a given mRNA via the RNA interference (RNAi) pathway. In this mode of action one of the two strands of the duplex acts as a guide (antisense) strand, while the other (sense or passenger strand) has no direct function in gene silencing. It has been shown in the past that chemical modiﬁcation of oligonucleotides can improve their therapeutic potential (4). The nature of the chemical modiﬁcation has to be carefully tuned with regard to the envisaged gene-silencing mechanism. For single-stranded antisense oligonucleo- tides acting either via a steric block or an RNaseH- dependent mechanism, high afﬁnity to the RNA target besides improved biostability and efﬁcient cellular uptake are required. These features are to some extent attainable by sugar/backbone modiﬁcation. Successful candidates of such chemically modiﬁed oligonucleotide analogs include HNA (5), locked nucleic acid (LNA) (6,7), 20-MOE-RNA and related 20-O-modiﬁed RNA (8), PNA and modiﬁcations thereof (9), Morpholino-NAs (10) and tricyclo-DNA (11,12). For siRNA-mediated mechanisms on the other hand, homogeneously increased stability of chemically modiﬁed oligoribonucleotides to their mRNA target may confer adverse thermodynamic effects on guide strand dis- sociation compared with target RNA binding. In addition, guide strand selection by RNA-induced silencing complex (RISC) exploits a thermodynamic asymmetry of base pairing at the two ends of the siRNA duplex, favoring that strand as a guide strand which shows weaker base pairing at its 50-end (13,14). This highlights the subtle interplay between target afﬁnity and RNAi activity and poses a challenge for the optimal design of an siRNA duplex. To remedy these drawbacks, several approaches have been evaluated in the recent past. For example, the thermodynamic asymmetry has been exploited to favor *To whom correspondence should be addressed. Tel: +41(0)31 631-4355; Fax: +41(0)31 631-3422; Email: leumann@ioc.unibe.ch The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.