Design and Synthesis of Optimized LNA Oligonucleotides

The YNRA4 fragment (32 nucleotides) was tiled with 16 bp long complementary nucleotides resulting in 17 possible designs. We mapped the full set of antisense oligonucleotides to the human transcriptome (Ensembl v84) and miRBase. Oligonucleotides with no off-targets when 3 mismatches are allowed were retained. Of the retained LNAs, we chose the oligonucleotide with the highest melting temperature (Tm). The resulting fully modified LNA (ACCCACTACCATCGGA, targeting TCCGATGGTAGTGGGT) has a Tm of 89.9 °C. In addition to the fully LNA-modified oligo, for the same sequence we ordered 2′-O-methyl and 2′-methoxy-ethoxy modified nucleotides and half modified (alternating modified – non-modified nucleotides) oligos at Integrated DNA Technologies. Sequences are available in Supplemental Table 1.