As the most stringent test, we first performed unbiased MD simulations starting from an extended linear conformation to assess the ability of MccJ25 to fold with minimal bias on the preferred structures. No pre-lasso structures were observed in 7.2 µs, but pre-tadpoles and premacrocycles (defined by distances of 6 Å or less between Gly1-Gly21) were detected, representing ~3% (21,288 structures) of the sampled phase space. The DBSCAN cluster analysis identified four structural clusters (Figure ). Two clusters, 1 and 2, contained premacrocycles without the characteristic β-hairpin secondary structure that is unique to MccJ25, including some with a short alpha-helix (cluster 2). The other two clusters, 0 and 3, contained pre-tadpole structures with the β-hairpin secondary structure, indicating the formation of this structure is readily accessible in unbiased simulations when starting from an extended conformation. This antiparallel beta structure is formed by hydrogen bonds between residues 7-16. Thus, while lasso folding and cyclization are enzyme-stabilized in nature, the β-hairpin formation is accessible in the absence of enzymes, suggesting a potential pathway for de novo pre-lasso folding.