Fine-Tuning of ENA®Gapmers as Antisense Oligonucleotides for Sequence-Specific Inhibition

Abstract

For gene validation and the development of oligonucleotide agents, 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) antisense gapmers are widely available. An in vitro Escherichia coli RNase H reaction analysis using ENA gapmers and an RNA oligonucleotide with mouse peptidylarginine deiminase 4 (PADI4) gene sequences revealed that the RNA oligonucleotide was specifically cleaved in the only reported case of the use of an ENA gapmer with an antisense sequence. On the other hand, duplexes of the full-length transcripts of PADI4 mRNA and ENA gapmers with a wide DNA window were cleaved not only at the target site, but also at nontarget sites by RNase H derived from partial base-pairing between the transcript and the ENA gapmer. When the DNA window region of the ENA gapmer was shortened to 5 or 6 nucleotides, the nontarget cleavage was effectively diminished. Moreover, the specific inhibition of PADI4 mRNA expression was observed in the cotransfection of PADI4 cDNA and ENA gapmers containing a short DNA region into NIH3T3 cells. These results demonstrated that ENA gapmers with a short DNA region improved the sequence-specificity of mRNA downregulation. These optimized ENA gapmers could reduce the "off-target" effect and be applicable to gene validation and oligonucleotide therapeutics.