Positional and Neighboring Base Pair Effects on the Thermodynamic Stability of RNA Single Mismatches

The known functions and roles of RNA in nature are vast. Similarly, the types of secondary structure motifs present in RNA are also diverse. These include canonical helices and non-canonical regions, such as internal, bulge, hairpin, and multi-branch loops. Single mismatches, or 1×1 internal loops, are the most frequently occurring secondary structure motif in ribosomal RNA (1) and often times serve integral structural and/or functional roles (2–12). Consequently, single mismatches have been utilized in therapeutic techniques as a target (13–16), an aptamer drug (17, 18), and a probe (19–22). One example of a therapeutic technique utilizing this secondary structure motif is demonstrated by recent studies examining the positional effect of single mismatches on the efficacy of RNA interference (RNAi) activity by placing mismatches at the center and the 5′ and 3′ ends of the sense stranded-small interfering RNA (ss-siRNA) component (19–22). siRNA duplexes with single mismatches placed at the 3′ terminus of the sense strand showed increased RNAi activity when compared to perfectly matched siRNA duplexes or those containing mismatches at the center or 5′ end. These enhanced siRNAs are known as ‘fork-siRNA duplexes’ (19, 22). Furthermore, the activity of short hairpin RNAs (shRNAs) has also been shown to be increased by the incorporation of 3′ terminal single mismatches and a decreased overall thermodynamic stability (ΔG). Westerhout and Berkhout further demonstrated shRNAs were most effective if they possessed a free energy value within a defined window, while also containing 3′ terminal mismatches (21). Synthetic fork-siRNAs and shRNAs are effective therapeutics to suppress gene expression by interacting with the RNA-induced silencing complexes (RISCs) and thereby invoking sequence-specific RNAi activity. 3′ terminal mismatches allow for recognition and duplex unwinding by the RISC helicase activity (23–27). It has been proposed they also minimize off-target gene silencing by resulting in direction specific disassociation of the siRNA and act as sequence specific RNAi mediators in RISC (19). The algorithms most commonly used to predict secondary structure from sequence are based on free energy minimization (28–34) using nearest neighbor parameters and have been incorporated into user-friendly, computer programs. In this method, a given sequence is folded into possible conformations. The total free energy values for each conformation are calculated by summing together the free energy parameters of all secondary structure motifs (experimental or predicted). This results in an optimal structure and a series of suboptimal structures. The optimal structure has the lowest free energy and is predicted to be the predominate structure in solution. These prediction algorithms utilize two methods when assigning free energy parameters to non-canonical regions. If thermodynamic parameters for a given motif are available, the experimentally determined free energy value is assigned. If such parameters have not been experimentally determined, a predicted free energy value is assigned. Much work has been done to thermodynamically characterize single mismatches placed in the center of a duplex (1, 35–37). These studies have shown the contribution of single mismatches to duplex thermodynamics to be dependent on the identity of the nearest neighbors and the identity of the mismatched nucleotides (1, 35–37). For example, we (36) recently proposed a single mismatch specific algorithm which utilizes three parameters consisting of a total of nine variables. The free energy of an RNA duplex containing a single mismatch which has not been thermodynamically characterized can be calculated by: ΔG°37,singlemismatch=ΔG°37,mismatchnt+ΔG°37,mismatch−NNinteraction+ΔG°37,AU+ΔG°37,GU (1) Here, ΔG°37,mismatch nt is −0.3, −2.1, and −0.6 kcal/mol for A·G, G·G, and U·U mismatches, respectively; ΔG°37,mismatch-NN interaction is 0.6, 0.0, 0.6, −0.5, and −0.9 kcal/mol for [Y5′R_R3′R3′R_Y5′],[R5′Y_Y3′Y3′Y_R5′],[Y5′Y_R3′R3′Y_Y5′],[Y5′R_Y3′R3′Y_R5′], and [R5′R_Y3′Y3′Y_R5′] mismatch and nearest neighbor combinations, respectively, when A and G are categorized as purines (R) and C and U are categorized as pyrimidines (Y); ΔG°37,AU is a penalty of 1.1 kcal/mol for replacing a G-C closing base pair with an A-U base pair; and ΔG°37,GU is a penalty of 1.4 kcal/mol for replacing a G-C closing base pair with a G-U base pair. All other combinations of single mismatch nucleotides and nearest neighbors are assumed to contribute no favorable or unfavorable contributions to duplex stability and are assigned a free energy value of zero (36). In addition to the identity of the nearest neighbors and mismatched nucleotides, it is important to note studies have reported the dependence of the thermodynamic stability of small RNA motifs on the duplex position and the identity of non-nearest neighbors (37–42). An example of the thermodynamic dependence on the motif’s duplex position was demonstrated by Kierzek and coworkers investigating the thermodynamics of single mismatches (37). A·A and U·U single mismatches had increased stability the closer they were placed towards the end of the duplex, while G·G single mismatches were unaffected by position in the duplex (37). An investigation of bulges of one nucleotide (38) demonstrated thermodynamic dependence on the identity of non-nearest neighbors and further showed a clear and direct relationship between the thermodynamic stability of the parental duplex and the thermodynamic contribution of the bulge. For example, the bulge [G5′C_U3′C3′A5′] was placed in the center of two different duplex sequences and a 3.0 kcal/mol difference in free energy contribution between the two duplexes was obtained (38). Similarly, a recent thermodynamic study on 1×2 loops (43) showed a strong dependence on the identity of non-nearest neighbors. Placing the 1 × 2 loop [C5′C_C3′G3′AU_G5′] in the center of two different duplex sequences resulted in a difference in free energy contribution of 2.8 kcal/mol (43, 44). Additionally, for tetraloops, or hairpins of four, non-nearest neighbor effects were observed when comparing the thermodynamics of tetraloop contribution to duplex stability when placed in the sequences 5′GCCNNNNGGC3′ and 5′GGCNNNNGCC3′. When tetraloops are placed in the latter stem sequence, they were, on average, 0.6 kcal/mol more stable than in the former sequence (44). Because current secondary structure prediction algorithms assume the thermodynamic contribution of small RNA motifs is independent of both its position in the duplex and identity of the non-nearest neighbors, these results suggest a better understanding of positional and non-nearest neighbor effects may lead to improved algorithms to predict secondary structure from sequence. This work investigates the positional and non-nearest neighbor effects on the thermodynamic contribution of single mismatches by thermodynamically characterizing the same single mismatch-nearest neighbor combinations at three duplex positions within the same stem. Results show positional and/or non-nearest neighbor effects play a role in defining the thermodynamic contribution of single mismatches to duplex stability.