7000 Nucleic Acids Research, 2007, Vol. 35, No. 20 Figure 4. Temperature dependence of the hairpin energy landscape. The PMF of the hairpin is plotted as a function of hairpin RMSD (compared of crystal conformer) versus the number of non-native hydrogen bonds (NN) at 275, 300, 347 and 400 K. Local minima are considered to be signiﬁcant when they contains more than 2% of the population and are separated by a barrier of at least 1.5 kcal/mol. All RMSD values are in Angstroms units (A˚ ). PMF is plotted in units of kcal/mol, where 1 kcal roughly equals to 0.6 KT at 300 K. DISCUSSION Due to the ruggedness of the energy landscape typically associated with RNA (58,59), RNA is prone to be trapped in meta-stable local minima. Recent kinetics data sug- gested that even the folding of a small 8-mer RNA hairpin consisting of a tetraloop and 2-bp long stem has a rugged energy landscape and requires a 4-state model to accurately describe its folding (36). In addition, several computational studies on RNA tetraloop hairpin have yield alternate 3-state folding models and competing folding mechanism referred to as expansion, unzipping and a combination of both (38,40). Here, we employed replica exchange algorithm in addition to standard molecular dynamics techniques during our simulations in order to enhance sampling. Characterization of the T-loop motif and its structural components (U-turn and U:A trans W/H bp) in diﬀerent structural contexts reveals that the T-loop itself, and especially the U-turn sub-motif, is an intrinsically ﬂexible motif. Its sequence does not have a tendency to fold into conformations commonly associated with this motif in the ribosome and other naturally occurring RNAs. This is supported by the observation that the T-loop is usually found to participate in tertiary structure formation through long-range interactions (Verzemnieks and Jaeger, manuscript in preparation). While the U-turn sub-motif exhibits high conformational ﬂexibility regardless of the helical stem, the U:A trans W/H bp, on the other hand, is more stable in the presence of a hairpin stem. It seems that although the T-loop can be considered as a 5-nte motif composed only of the U-turn and the U:A trans W/H bp, the stability of the T-loop is indeed enhanced by the stacking of an additional WC bp on the U:A trans W/H bp as found in the UA-handle (Verzemnieks and Jaeger, manuscript in preparation). In the scenario where this additional WC base pair is missing, the stability of the T-loop motif is probably more dependent on the structural context of the RNA and the availability of tertiary interactions. For example, the T-loop has been found to fold in the presence of another T-loop by providing intercalating bases to each other as seen in the S-domain of Rnase P and domain 1 of the 23S ribosomal RNA (34). We speculate that the sequence information embedded in the U-turn sub-motif and T-loop are likely to be important during the later conformational search stage that eventually leads to native helix packing. Our results imply that the U-turn probably does not play an active role during the secondary structure formation despite its frequent occurrence. We speculate that while these ‘special’ terminal loops might stabilize the hairpin once it has formed, they do not necessarily contribute to the folding of the hairpin. This result reinforces the observa- tion by Pleij and coworkers that hairpin folding kinetics are the same when a GNRA tetraloop is substituted by a non-GNRA tetraloop or when the loop size is increased from 4 to 6 nt (42). Comparable studies of the T-loop with a more stable UA-handle (Verzemnieks and Jaeger, manuscript in preparation) might be interesting for future investigation. The partially zipped hairpin-like transition state found in this study leads us to believe that zipping is extremely rapid and partially zipped structures are only short-lived transient species for a small hairpin. The transition state described above may be a feature of small hairpins whose stems length does not exceed a few nucleotides. As the stem lengthens, complete zipping might not be as rapid, however. It is interesting to note that Gruebele and coworkers (36) observed unusual folding kinetics for a small UNCG tetraloop hairpin that were suggestive of an underlying rugged energy landscape. Our observation of multiple minima at diﬀerent temperatures in the PMFs of the T-loop hairpin seems to corroborate their data (36). Although the actual structure and the nature of the minima may diﬀer from case to case depending on the RNA system being studied (e.g. UNCG tetraloop hairpin versus the T-loop hairpin), we believe such phenomenon reﬂects the intrinsic ruggedness of the RNA energy landscape and it is a generic feature shared by many RNA molecules.