However, the abiotic folding of lasso peptides de novo is underexplored. This work uses extensive MD simulations to revisit the folding propensity of MccJ25, which was previously suggested to rapidly transition to the unnatural left-handed structure. We find that improved force fields support the preference for the natural right-handed conformation and demonstrate increased access to the pre-lasso ensemble. For MccJ25, the observed preference for a righthanded pre-lasso may be the consequence of some, or all of the L-configured residues, that predispose the peptide to favor the right-handed structure. This right-handed predisposition, consistent with other natural lasso peptides, may be reinforced upon binding and cyclization as mediated by the lasso cyclase enzyme. Additional studies examining the effect of Dconfigured residue substitutions on pre-lasso handedness preference are needed. We present new tools employing triangulation and the barycentric transformation to definitively characterize pre-lasso conformations and distinguish handedness. These tools for characterizing pre-lasso conformations could be used together with computational tools like LassoHTP, which predicts bonded lasso peptide structures and ensembles, 56 in future design strategies for lasso peptide diversification. We also demonstrate that chemical modification the ring-forming residues increases pre-lasso stabilization through π-π stacking and sulfur interactions. Similarly, His5 protonation further increases the pre-lasso ensemble stability. In this case, the increased hydrophilicity of imidazolium-bearing His5 reorients the backbone, enabling hydrogen bonding with the C-terminal Gly1 carboxylate. Importantly, our findings demonstrate that alteration of the sequence, incorporating unnatural modifications and ionizable functionalities could permit a rational design of lasso peptides. These developments mark the first steps toward realizing a generalizable platform for the chemical synthesis of lassos peptides, which could permit access to mirror-image lassos (from D-amino acids), and the incorporation of non-canonical residues, including D-amino acids, β-amino acids, peptoids, and others, to compliment biosynthetic approaches that diversify these remarkable scaffolds toward the design of novel therapeutics.