Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters

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 progesterone 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 2′F-RNA, 2′OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modification: 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 demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.

Activation of LDL Receptor Expression by Small RNAs Complementary to a Noncoding Transcript that Overlaps the LDLR Promoter

“…When variants of LDLR-28 were tested, activation was observed regardless of whether the 2′-O-methyl or 2′-fluoro modifications were on the sense or antisense strand. The phenomenon that similar patterns of chemical modification have different effects on gene activation when applied to different sequences has been observed previously in chemically modified agRNAs that activate PR expression (Watts et al, 2010b). …”

“…Several reports have also appeared suggesting that small RNAs complementary to gene promoters can also regulate gene expression. These antigene RNAs (agRNAs) (we use this terminology to distinguish them from RNAs complementary to mRNA) can either inhibit (Morris et al, 2004; Ting et al, 2005; Janowski et al, 2005; Janowski et al, 2006; Kim et al, 2006; Han et al, 2007; Janowski et al, 2007; Napoli et al 2009; Hawkins, 2009; Watts et al 2010b; Yue et al, 2010) or activate gene expression (Li et al, 2006; Janowski et al, 2007; Morris et al, 2008; Place et al, 2008; Huang et al, 2010; Watts et al, 2010b; Yue et al, 2010). Argonaute 2 (AGO2), a key protein involved in RNAi (Siomi and Siomi, 2009), has been reported to be required for gene activation (Li et al, 2006; Morris et al, 2008; Chu et al, 2010), and both AGO2 and a related protein, AGO1, have been implicated in transcriptional silencing (Janowski et al, 2006; Kim et al, 2006; Napoli et al, 2009; Chu et al, 2010).…”

The role of antisense long noncoding RNA in small RNA-triggered gene activation

“…Similarly, small RNA-induced TGA has been reported in mammalian systems Watts et al 2010;Voutila et al 2012;Matsui et al 2013;Reebye et al 2013a,b), though the molecular mechanisms have not been fully elucidated. In these previous studies, TGA has been induced by two or more shRNAs that target the promoter sequence (Chu et al 2012).…”

Making sense of Dlx1 antisense RNA

“…Naturally, the approach to target any NAT of a gene is difficult, as it is extremely important not to disrupt the function of the gene itself so to avoid the misattribution of a phenotype as function of the lncRNA in question in its gene expression modulating function, development or pathogenesis. Several approaches have been taken to knockdown antisense RNA using siRNA technology (Janowski et al, 2007;Modarresi et al, 2012;Rapicavoli et al, 2011;Sheik Mohamed et al, 2010;Watts et al, 2010). Often though, the siRNA knockdown methodology suffers from off-target side effects, impacting on the interpretation of the resulting phenotype (Clark et al, 2008;Harborth et al, 2003;Scacheri et al, 2004;Sioud, 2011).…”