5242–5259 Nucleic Acids Research, 2010, Vol. 38, No. 15 doi:10.1093/nar/gkq258 Published online 19 April 2010 Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters Jonathan K. Watts1, Dongbo Yu1, Klaus Charisse2, Christophe Montaillier3, Pierre Potier3, Muthiah Manoharan2 and David R. Corey1,* 1Department of Pharmacology and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, 75390, 2Alnylam Pharmaceuticals, Cambridge, MA, 02142, USA and 3SIGMA Custom Products, Genopole Campus 1, 5 rue Desbrue` res, 91030 Evry Cedex, France Received October 14, 2009; Revised March 19, 2010; Accepted March 29, 2010 ABSTRACT Antigene RNAs (agRNAs) are small RNA duplexes that target non-coding transcripts rather than mRNA and specifically suppress or activate gene expression in a sequence-dependent manner. For many applications in vivo, it is likely that agRNAs will require chemical modification. We have synthesized agRNAs that contain different classes of chemical modification and have tested their ability to modulate expression of the human proges- terone receptor gene. We find that both silencing and activating agRNAs can retain activity after modification. Both guide and passenger strands can be modified and functional agRNAs can contain 20F-RNA, 20OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modifica- tion: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded. These data demon- strate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications. INTRODUCTION The therapeutic potential of gene silencing by small interfering RNAs (siRNAs) is widely recognized (1). siRNA clinical candidates are commonly modiﬁed to improve their stability, speciﬁcity and potency (2–4). Modiﬁed siRNAs have been shown to reduce oﬀ-target eﬀects resulting from the miRNA pathway (5,6), the innate immune system (7,8) and loading of the wrong strand (6,9,10). In addition, chemically modiﬁed siRNAs have been used to deepen our understanding of the mech- anism of RNAi (11–16) and improve siRNA delivery (17,18). Antigene RNAs (agRNAs) are small duplex RNAs that target gene promoters. Like siRNAs, they are generally 19 bp with 2-nt overhangs at the 30-ends. Depending on target sequence, cell type and basal level of expression, agRNAs can either silence (19–21) or activate (22–24) gene expression at the transcriptional level. Like gene silencing by siRNAs, modulation of gene expression by agRNAs involves argonaute (AGO) proteins (25,26). However, instead of targeting mRNA, agRNAs target non-coding RNA transcripts (ncRNAs) overlapping gene promoters (27,28). While agRNAs can target either sense or antisense non-coding transcripts, the agRNAs discussed in this study target an antisense transcript overlapping the promoter of the progesterone receptor (PR) gene (28). Thus the guide strand of these agRNA duplexes is the sense strand, in contrast to siRNAs, for which the guide strand is by deﬁnition antisense (i.e. com- plementary to the mRNA). agRNAs that activate gene expression could be used to modulate expression of disease-associated genes whose upregulation might be beneﬁcial. As with current gene silencing technology, however, it is likely that any devel- opment of agRNAs for in vivo use will require chemical modiﬁcation to improve stability and biodistribution. In this study we examine the eﬀects of modiﬁed *To whom correspondence should be addressed. Tel +1 214 645 6155; Fax +1 214 645 6067; Email: david.corey@utsouthwestern.edu 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.