*3.2.1 Prediction of secondary structure*

A sense oligonucleotide should harbor an overlapping sequence of an mRNA-AS transcript interaction, i.e., a single-stranded loop or bulge [18, 20]. The single-stranded loops are the potential sites of mRNA-AS transcript interactions. To seek the single-stranded regions of an mRNA where the relevant AS transcript interacts with, secondary structures of mRNA (especially, 3′UTR) were predicted using the mfold program [29]. Other prediction programs can be used. Regions conserved among predicted mRNA (especially 3′UTR) structures are selected, and candidates of several sense oligonucleotides are designed from the stem-loop regions.

Generally, it is unnecessary to predict the secondary structures of AS transcripts. As mentioned above, the overlapping sequence of an AS transcript is complementary to that of the corresponding mRNA. Therefore, the secondary structures of the AS transcript are a mirror image of the mRNA. The stems and loops of the AS transcript are formed at the same positions as the mRNA.

### *3.2.2 Design of sense oligonucleotides*

The sense oligonucleotide consists of either synthetic oligodeoxyribonucleotide (DNA) or synthetic oligoribonucleotides (RNA) with modifications of the oligonucleotide backbone, sugars, bases, and the 5′-phosphate (described later). The sequences of sense oligonucleotides (about 20 nt long in our cases) designed according to the mRNA sequence included at least one single-stranded loop in the conserved region [14, 19]. From the sequences of sense oligonucleotides, the elements that may provoke innate immunity responses through Toll-like receptors (TLR3, 7, 8, and 9) should be eliminated, such as GU-rich motifs (e.g., 5′-GUGU-3′), CG, GGG, GGGG, and CCCC [14, 19, 30]. It is possible that some oligonucleotides show off-target effects, even after the exclusion of these motifs. To attain the specificity of a target mRNA and avoid off-target effects, homology search in the DDBJ/EMBL/GenBank databases should be performed. Trials and errors are necessary to select effective sense oligonucleotides among several candidates.

Note that not all the candidate sense oligonucleotides reduce the levels of specific mRNA species, whereas some oligonucleotides increase the mRNA levels. As abovementioned, AS transcripts modulate the expression of each gene either positively or negatively; the AS transcript stabilizes mRNA (**Figures 3** and **4**) or destabilizes mRNA (**Figure 5**).

Changes in mRNA levels also depend on the region of the mRNA-AS transcript interactions [14, 24]. Six sense oligonucleotides to the 3′UTR of the rat *Tnf* mRNA were designed to the regions whose secondary structures were conserved [24]. Among them, only one sense oligonucleotide decreased *Tnf* mRNA levels, four increased, and one did not alter. RNA-binding proteins, e.g., HuR and TTP, may change the effect of each sense oligonucleotide.

Additionally, both several RNA-binding proteins and microRNAs control the mRNA stability (see Section 2.2). Therefore, it is difficult to predict whether knockdown of AS transcript using a sense oligonucleotide causes either an increase or decrease in mRNA levels (see also Section 3.3).

### *3.2.3 Negative controls of sense oligonucleotides*

Several types of oligonucleotides are frequently used as negative controls. A negative control that is suitable for your experiments should be selected because not all the negative controls work well in the experiments.
