Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Abstract

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson-Crick nearestneighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson-Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson-Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3 ′ or 5 ′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.

Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

“…The average deviations in thermodynamic parameter values for the hairpins with group I bulge loops or fully base-paired stems are 4.8, 13.3, and 0.3 for ΔH°, ΔS°, DG°3 7 , respectively. The average errors for the hairpins with group II bulge loops are larger (7.6, 24.2, and 0.4 for ΔH°, ΔS°, DG°3 7 , respectively) than for the fully base-paired or group I bulge loops perhaps indicative of some slight non-two-state melting (Kent et al 2014). …”

“…The insertion of the group I bulge (A or 2-AP) decreases the stability of the hairpin by on average 0.4 kcal/mol. We previously developed a model to predict the influence of single nucleotide bulge loops on the stability of duplex and hairpin formation (Kent et al 2014). That relationship is given in the equation below:…”

“…For hairpins with embedded bulge loops, the DG°3 7stem is calculated for the distal stem (Lim et al 2012;Kent et al 2014). Note that with this model, the bulged nucleotide does not disrupt the nearest-neighbor interactions of the base pairs neighboring the bulge loop.…”

“…The current work is a continuation of our studies on the stability and structure of bulge loops in the context of an RNA hairpin stem (Lim et al 2012;Kent et al 2014). A simple model has been proposed to predict the influence of bulge loops on the stability of hairpin formation that depends upon non-nearest-neighbor interactions (stability of the stem distal to the hairpin loop) (Kent et al 2014).…”

“…A simple model has been proposed to predict the influence of bulge loops on the stability of hairpin formation that depends upon non-nearest-neighbor interactions (stability of the stem distal to the hairpin loop) (Kent et al 2014). In-line structure probing has been used to determine the identity of the bulge in cases where there was ambiguity (group II or III bulge loops).…”