Design of Antisense Oligonucleotides

The target N-gene sequence of SARS-CoV-2, as mentioned in the Supporting Information, was supplied to software, Soligo,⁶³ for statistical folding of nucleic acids and studies of regulatory RNAs. The ASOs were predicted to maintain the folding temperature as 37 °C and ionic conditions of 1 M sodium chloride for a preferred length of ASO as 20 nucleotide bases. The filter criteria were set as follows: (1) The GC% will be within 40% to 60%; (2) the target sequences with GGGG will be eliminated; (3) the average unpaired probability of the ASOs should be within ≥0.5 for target site nucleotides; (4) among sites satisfying criteria 1–3, the top 20 will be considered with the highest average unpaired probability. In order to reduce the number of reported sites, the average unpaired probability was also used in filter criteria 3 and 4. The disruption energy calculation in the Web servers was also optimized accordingly. Finally, the binding energies of the ASOs were also compared with the target sequence to decide on the sequences.