The pbuE adenine riboswitch undergoes metal ion-dependent folding that involves a loop-loop interaction. Binding of 2-aminopurine to the aptamer domain strongly correlates with the ability of the loops to interact, and single-molecule FRET studies reveal that folding proceeds via a discrete intermediate. Folding occurs in the absence of adenine ligand, but ligand binding stabilizes the folded structure by increasing the folding rate and decreasing the unfolding rate, and it lowers the magnesium ion concentration required to promote the loop-loop interaction. Individual aptamer molecules exhibit great heterogeneity in folding and unfolding rates, but this is reduced in the presence of adenine. In the full riboswitch, the adenine binding domain fails to fold because of conformational competition by the terminator stem. Thus, riboswitch function should depend on the relative rates of ligand binding and the transcriptional process.

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

U sually caused by progressive atherosclerosis and associated thrombosis, peripheral arterial disease (PAD) is characterized by a reduced blood flow to the lower limbs, leading to ischemia during physical exertion and intermittent claudication as clinical presentation. 1 Intermittent claudication, classically defined by pain and muscle cramps when walking, results in a greatly reduced walking capacity and functional status. 2,3 This functional decline lowers patient quality of life and willingness to engage in physical activity, which further worsens the risk of cardiovascular disease 4 and premature mortality. 5 For these reasons, improving walking capacity is now considered a priority for the treatment of PAD. 6 Aerobic exercise, typically in the form of walking, is considered a first-line treatment in this population. 7 Scientific evidence supports the existing guidelines that recommend ≥3/wk of supervised exercise sessions of intermittent walking, according to the pain threshold (3-4/5), for 30-45 min/ session, for ≥3 mo. [7][8][9] Recent meta-analyses and clinical trials have shown an increase in maximum walking distance (MWD) on treadmill tests after supervised exercise therapy (SET) in patients with symptomatic PAD. [10][11][12][13] Although home-based exercise has shown encouraging results, the magnitude of these results was inferior to SET. 14 According to the American Heart Association, the optimal SET modality still needs to be determined for patients with PAD. 15 Accumulating data indicate that other types of SET, such as Nordic walking, underwater exercise, and resistance training, could also be effective in improving walking capacity in patients with PAD. [16][17][18][19] Recently, a meta-analysis 8 suggested that other modes of exercise (without any distinction between them) could improve walking ability similar to supervised intermittent walking. Although this