Antiviral drug discovery continues to be an essential complement to vaccine development for overcoming the global pandemic caused by SARS-CoV-2. The genomic RNA of SARS-CoV-2 contains structural elements important for viral replication and/or pathogenesis making them potential therapeutic targets. Here we report on the stem-loop II motif, a highly conserved noncoding RNA element. Based on our homology model we determined that the G to U transversion in the SARS-CoV-2 stem-loop II motif (S2MG35U) forms a C-U base-pair isosteric to the C-G base-pair in the early 2000's SARS-CoV (S2M). In addition, chemo-enzymatic probing and molecular dynamics simulations indicate the S2MG35U conformational profile is altered compared to S2M in the apical loop region. We explored S2MG35U as a potential drug target by docking a library of FDA approved drugs. Enzymatic probing of the best docking ligands (aminoglycosides and polymyxins) indicated that polymyxin binding alters the conformational profile and/or secondary structure of the RNA. The SARS-CoV-2 stem-loop II motif conformational differences due to nucleotide transversion and ligand binding are highly significant and provide insight for future drug discovery efforts since the conformation of noncoding RNA elements affects their function. (c) 2021 Elsevier Inc. All rights reserved.