Remarkable stability of hairpins containing 2′,5′-linked RNA loops

We report here the results of a comparative study of hairpin loops that differ in the connectivity of phosphodiester linkages (3',5'- versus 2',5'-linkages). In addition, we have studied the effect of changing the stem composition on the thermodynamic stability of hairpin loops. Specifically, we constructed hairpins containing one of six stem duplex combinations. i.e., DNA:DNA ("DD"), RNA:RNA ("RR"), DNA:RNA ("DR"), 2',5'-RNA:RNA ("(R) under barR"), 2',5'-RNA:DNA ("(R) under barD"), and 2',5'-RNA:2',5'-RNA ("(RR) under bar"), and one of three tetraloop compositions, i.e., 5'-RNA ("(R) under bar"), RNA ("R"), and DNA ("D"). All hairpins contained the conserved and well-studied loop sequence 5'-...C(UUCG)G...-3' [Cheong et al. Nature 1990, 346, 680-682]. We show that the 2',5'-linked loop C((UUCG) under bar )G. i.e., ... C(3'p5')U(2'p5')U(2'p5')C(2'p5)G(2'p5')G(3'p5),..., like its "normal" RNA counterpart, forms an unusually stable tetraloop structure. We also show that the stability imparted by 2',5'-RNA loops is dependent on base sequence, a property that is shared with the regioisomeric 3',5'-RNA loops. Remarkably, we find that the stability of the (UUCG) under bar tetraloop is virtually independent of the hairpin stem composition (DD, RR, (RR) under bar, etc.), whereas the native RNA tetraloop exerts extra stability only when the stem is duplex RNA (R:R)-As a result, the relative stabilities of hairpins with a 2',5'-linked tetraloop, e.g. ggac((UUCG) under bar )gtcc (T-m = 61.4 degreesC), are often superior to those with RNA tetraloops, e.g. ggac(UUCG)gtcc (T-m = 54.6 degreesC). In fact, it has been possible to observe the formation of a 2',5'-RNA:DNA hybrid duplex by linking the hybrid's strands to a ((UUCG) under bar) loop, These duplexes ((R) under barD), which are not stable enough to form in an intermolecular complex [Wasner et al. Biochemistry 1998, 37, 7478-7486], were stable at room temperature (T-m similar to 50 degreesC). Thus, 2',5'-loops have potentially important implications in the study of nucleic acid complexes where structural data are not yet available. Furthermore, they may be particularly useful as structural motifs for synthetic ribozymes and nucleic acid "aptamers".