Published online 16 October 2007 Nucleic Acids Research, 2007, Vol. 35, No. 20 6995–7002 doi:10.1093/nar/gkm719 Probing the structural hierarchy and energy landscape of an RNA T-loop hairpin Zhuoyun Zhuang 1 , Luc Jaeger1,2 and Joan-Emma Shea1,* 1Department of Chemistry and Biochemistry and 2Bio-molecular Science and Engineering Program, University of California, Santa Barbara, CA 93106-9510, USA Received July 10, 2007; Revised and Accepted August 29, 2007 ABSTRACT The T-loop motif is an important recurrent RNA structural building block consisting of a U-turn sub- motif and a UA trans Watson–Crick/Hoogsteen base pair. In the presence of a hairpin stem, the UA non- canonical base pair becomes part of the UA-handle motif. To probe the hierarchical organization and energy landscape of the T-loop, we performed replica exchange molecular dynamics (REMD) simu- lations of the T-loop in isolation and as part of a hairpin. Our simulations reveal that the isolated T-loop adopts coil conformers stabilized by base stacking. The T-loop hairpin shows a highly rugged energy landscape featuring multiple local minima with a transition state for folding consisting of partially zipped states. The U-turn displays a high conformational flexibility both when the T-loop is in isolation and as part of a hairpin. On the other hand, the stability of the UA non-canonical base pair is enhanced in the presence of the UA-handle. This motif is apparently a key component for stabilizing the T-loop, while the U-turn is mostly involved in long-range interaction. Our results suggest that the stability and folding of small RNA motifs are highly dependent on local context. INTRODUCTION The folding of RNA is considered to be hierarchically organized (1–10). Four distinct stages can be distin- guished: secondary structure formation, collapse mediated by metal ion condensation (11–14), partial helix assembly based on limited number of native tertiary interactions and conformational search leading to the ﬁnal native structure. The last step (conformational search) is highly sequence dependent and requires the formation of recurrent structural motifs, small sets of nucleotides that code for speciﬁc folds. A powerful approach to solving the RNA folding problem lies in studying these recurrent structural motifs, to establish (i) how these motifs fold as independent units and (ii) how these units further assemble and guide the folding of the larger RNA molecule. The RNA hairpin motif is one of the most common recurrent secondary structure elements found in naturally occurring RNA. Hairpin motifs generally contain a loop 4–7 nt long (15), with the UNCG or GNRA tetraloops (N = any nucleotide; R = purine) and the T-loop occur- ring most frequently (15–20). These loops share a number of similarities including a non-Watson–Crick closing base pair (bp) and the reversal of the direction of the RNA backbone. For tetraloop and T-loop motifs, this is accomplished by the U-turn sub-motif of speciﬁc sequence signature 50-(g/u)NR-30 (17,21). Many hairpins in RNA are thermostable (22,23) and are involved in tertiary structure formation (15,24). GNRA and UNCG hairpins have been extensively studied and are known to fold autonomously in the absence of long-range tertiary interaction. It has been suggested that these hairpins contribute as nucleation sites for the folding of larger RNA sequences (25). NMR and X-ray studies of tetraloop hairpins in isolation (18,26–28) or within the context of larger RNA molecules (29–34) have shown well-deﬁned conformers with unique hydrogen bond networks in solution as well as within crystal packing. However, more recent experimental studies (18,25) on RNA tetra- loop hairpins in isolation suggest the existence of alternate loop conformations not previously observed by NMR or X-ray crystallography, intimating that local environment can modulate the conformations of these and other motifs. Additional recent studies using ultrafast femtosecond spectroscopy has probed similar alternate base stacking patterns in the GNRA tetraloop, further supporting the notion of dynamic, rather than static, RNA motifs (35). This work focuses on the folding of the T-loop, both in isolation and as part of a hairpin. Most experimental and theoretical studies focus on the more common GNRA and UNCG tetraloops (25,36–41), hence, our motivation to investigate alternate hairpin systems in an attempt to *To whom correspondence should be addressed. Tel: +1 805 893 5604; Fax: +1 805 893 4120; Email: shea@chem.ucsb.edu Correspondence may also be addressed to Luc Jaeger. Email: jaeger@chem.ucsb.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.