*3.2.3.4 Scrambled oligonucleotides and mock transfection*

A scrambled oligonucleotide is designed by base shuffling without changing the base composition of the relevant sense oligonucleotide [31]. Homology search screened by the BLAST program must eliminate candidates harboring unexpected homology to other mRNAs in the DDBJ/EMBL/GenBank databases.

When a sense oligonucleotide is introduced into cells using a transfection reagent, mock transfection is also necessary as a negative control of transfection. The mock transfection requires a transfection reagent alone, and an oligonucleotide is not introduced to the cells [18, 20].

### *3.2.4 Modification of sense oligonucleotides*

A variety of nucleases are present in the cells and blood, such as exonuclease, endonuclease, and ribonuclease (RNase) H1. RNase activity is very high in various cell lines, as well as blood and cells in many organs, including the liver. To protect sense oligonucleotides from these nucleases, phosphorothioate bonds and modified nucleic acids are commonly introduced to replace the phosphodiester bonds and (deoxy)ribose rings of native nucleotides, respectively [32, 33]. Indeed, *iNOS* sense oligonucleotides without modification did not reduce *iNOS* mRNA levels [18]. Locked nucleic acid (LNA) [34] and 2′-*O*-methyl nucleic acid (OmeNA) are frequently used as modified nucleic acids. In our cases, *iNOS* sense oligonucleotides were substituted with partial phosphorothioate bonds and LNAs or OmeNAs

*The Natural Antisense Transcript-Targeted Regulation Technology Using Sense Oligonucleotides… DOI: http://dx.doi.org/10.5772/intechopen.108281*

reduced the levels of *iNOS* mRNA and iNOS protein in hepatocytes [18, 19]. This is a critical point at which modifications are included to obtain effective sense oligonucleotides.
