In Fig. , we have shown some representative structures of the folded (F), misfolded (M) and unfolded (U) states with their corresponding CV values. As expected, the stem distance (along with end-to-end distance and Rg) increases from F→M→U as also evident from Figs . However, folded state is unique from the rest with a high number of hydrogen bonds and contacts between the stems of the tetraloop and a low value of ermsd. Both the misfolded and unfolded states have almost no contact and hydrogen bond within the stems. Misfolded states span a wide range of structures (due to broad minimum) differing mostly in terms of stem distance and end-to-end distance. The difference in the misfolded and unfolded state is mostly in the free energy values, which structurally correlates with stem distance, end-to-end distance, and Rg values. Therefore, beyond a certain stem distance, free energy rises fast and that we indicate here as the unfolded state. The free energy surfaces reweighted with respect to stem distance and other subsidiary collective variables are provided in the supplementary information (Fig. of SI). b) Influence of enantiomers in the folded state. The question that naturally comes is why L-arginine can't stabilize the folded state of GAAA tetraloop. First hypothesis was that interaction energy between RNA and stereoisomers are different. Therefore, we collected all the structures of the folded state (as defined earlier) and calculated the average number of hydrogen bond and average interaction energy between RNA and arginine. To our surprise, we find that both the quantities are same for L-and D-arginine, as shown in Fig. , ruling out any favorable interaction played by the D-arginine to stabilize the native folded state of GAAA tetraloop. Therefore, our next hypothesis was that RNA structures may be different in the folded state in presence of D-and Larginine (although they belong to the similar values of the CV), giving rise to similar interaction and hydrogen bond number. The reason for this hypothesis is that the nucleobases in the RNA form different types of interactions with respect to its complimentary/neighboring pair. These pairing schemes can be distinguished by the edge side of interaction forming the H-bonds. The possible edges of interactions are Watson-Crick edge, Hoogsteen/C-H edge and the Sugar edge. These base pairing schemes can be ideally achieved by cis/trans glycosidic rotation around the sugar backbone and the orientation of the H-bond formation between the nuclear bases. By identifying a given base, probable complementary base, and the type of glycosidic rotation, we can identify the ideal orientation of the state of a given residue of RNA. We have used the above-mentioned parameters based on the way portrayed by Leontis for geometric classification of RNA structures. As the stem region is expected to initiate the folding, we investigated the nature of base pairing in RNA and the glycosidic rotation around the sugar backbone for all the four base pairs in the stem region. A representation of various possible combinations of base pairing schemes is shown in the Fig. of SI. We have calculated the probability of the existence of these base paring orientations in RNA structures using Barnaba program. Each possible complementary interactions between the two bases in the RNA structure is labelled by a three-letter code. The first letter indicates the probable donating edge for base pairing in the 5' end stem region of RNA. The second letter defines the probable acceptor edge of interaction in the complimentary residue/neighboring residue in the RNA. The third letter indicate the possible cis/trans mode of interactions by glycosidic rotation of the sugar backbone. For example, WHc means that 5' end of a given stem residue with Watson-Crick edge is interacting with neighboring Hoogsteen acceptor edge at neighboring end in cis orientation. A representation of SHc and WCc state is shown in the Fig. and Fig. respectively. The details regarding the three-letter nomenclature are discussed in Table , and Fig. in SI section III. The probability of existence of the possible states at all four base pairs in the stem region is shown in the Fig. . The R1, R2, R3 and R4 indicate the base pairs in the stem region starting from the terminal end of the RNA. In all these base pairs, most tend to exist in WCc state (Watson-Crick edge, C-H edge, cis orientation). Next is the WWc base pairs, with much less probability. Figure shows that presence of D-or L-influence the geometry of only the terminal base pair (R1). While most of the structures preserve the WCc state in presence of D-arginine, there is a noticeable probability of forming SHc structures in presence of L-arginine. This indicates that the chirality effect is somehow present for the terminal base pair only. c) Single molecule binding studies. Now that we understand that chirality of the amino acids affects RNA structure in the folded state (especially for the terminal base pair), we wanted to further explore the cause for it. Therefore, we explored specific binding mode of the amino acids to the RNA by calculating the free energy of a single D-and L-arginine with RNA separately. For each system, we took the amino acid closest to R1 as the starting point and performed welltempered metadynamics simulations as a function of binding distance. Free energy surface for binding plotted with respect binding distance and angvec (see method) is shown in Fig. . and. Interestingly, we have observed a single minimum of lesser stability (-6.8 kcal/mol) in the bound state of for in L-arginine (Fig. ). The corresponding minima structure is shown in Fig. .