Influence of two bulge loops on the stability of RNA duplexes

Fifty-three RNA duplexes containing two single nucleotide bulge loops were optically melted in 1 M NaCl in order to determine the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each duplex. Because of the large number of possible combinations and lack of sequence effects observed previously, we limited our initial investigation to adenosine bulges, the most common naturally occurring bulge. For example, the following duplexes were investigated: 5′GGCAXYAGGC/3′CCG YX CCG, 5′GGCAXY GCC/3′CCG YXACGG, and 5′GGC XYAGCC/3′CCGAYX CGG. The identity of XY (where XY are Watson–Crick base pairs) and the total number of base pairs in the terminal and central stems were varied. As observed for duplexes with a single bulge loop, the effect of the two bulge loops on duplex stability is primarily influenced by non-nearest neighbor interactions. In particular, the stability of the stems influences the destabilization of the duplex by the inserted bulge loops. The model proposed to predict the influence of multiple bulge loops on duplex stability suggests that the destabilization of each bulge is related to the stability of the adjacent stems. A database of RNA secondary structures was examined to determine the naturally occurring abundance of duplexes containing multiple bulge loops. Of the 2000 examples found in the database, over 65% of the two bulge loops occur within 3 base pairs of each other. A database of RNA three-dimensional structures was examined to determine the structure of duplexes containing two single nucleotide bulge loops. The structures of the bulge loops are described.