A U⋅U Pair‐to‐U⋅C Pair Mutation‐Induced RNA Native Structure Destabilisation and Stretching‐Force‐Induced RNA Misfolding

Abstract

Little is known about how a non‐Watson–Crick pair affects the RNA folding dynamics. We studied the effects of a U⋅U‐to‐U⋅C pair mutation on the folding of a hairpin in human telomerase RNA. The ensemble thermal melting of the hairpins shows an on‐pathway intermediate with the disruption of the internal loop structure containing the U⋅U/U⋅C pairs. By using optical tweezers, we applied a stretching force on the terminal ends of the hairpins to probe directly the non‐nearest‐neighbour effects upon the mutations. The single U⋅U to U⋅C mutations are observed to 1) lower the mechanical unfolding force by approximately 1 picoNewton (pN) per mutation without affecting the unfolding reaction transition‐state position (thus suggesting that removing a single hydrogen bond affects the structural dynamics at least two base pairs away), 2) result in more frequent misfolding into a small hairpin at approximately 10 pN and 3) shift the folding reaction transition‐state position towards the native hairpin structure and slightly increase the mechanical folding kinetics (thus suggesting that untrapping from the misfolded state is not the rate‐limiting step).

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for −1 ribosomal frameshifting stimulation

“…1c). Thus, single-molecule mechanical unfolding studies can reveal the effect of subtle U to C substitution in a loop in a pseudoknot, as has been observed in RNA hairpins containing internal loops in the stems53. The relative mechanical stabilities (SF206 > SF220 > SF217) are consistent with the relative gel mobilities observed in the PAGE experiment (Fig.…”

Incorporating uracil and 5-halouracils into short peptide nucleic acids for enhanced recognition of A–U pairs in dsRNAs

“…It is interesting to note that singly-(at position 3, 5, or 6) and doubly-modified PNAs (at positions 3 and 5) generally show similar K d values at pH 7.5 (Table 2, Figure 4A, and Supplementary Figures S17 and S18), suggesting that the stabilizing effect of an internal X U modification may propagate beyond the nearest neighbor and the next nearest neighbor stacking partners in a triplex. Such non-nearest neighbor effect has been previously revealed for the formation of duplexes with other modifications and mutations by bulk thermal melting and single-molecule mechanical unfolding experiments (68)(69)(70)(71)(72)(73).…”

Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops

“…Data from optical melting experiments were fit using the linest function in Excel. The loops at pH 7 from this work were combined with all previous measurements of internal loop stabilities (Santa Lucia et al 1990Peritz et al 1991;Walter et al 1994;Wu et al 1995;Schroeder et al 1996;Xia et al 1997;Turner 2000, 2001;Burkard et al 2001;Chen et al 2004Chen et al , 2005Chen et al , 2009Bourdelat-Parks and Wartell 2005;Badhwar et al 2007;Znosko 2008, 2009;Hausmann and Znosko 2012;Zhong et al 2015). Thermodynamic data were manually curated, and data with nontwo-state behavior, highly probable alternate duplex structures, and three or more consecutive guanine nucleotides without singlestrand optical melting data were not included in the database for analysis.…”

“…All the thermodynamic data incorporated in the 2004 prediction model (Mathews et al 2004) were compiled and updated with new data for 126 RNA loops (Chen et al 2004(Chen et al , 2009Bourdelat-Parks and Wartell 2005;Badhwar et al 2007;Znosko 2008, 2009;Hausmann and Znosko 2012;Zhong et al 2015). A complete spreadsheet of loop thermodynamic parameters is available in the Supplemental Information.…”