Abstract
The thermodynamic stabilities of four natural prohead or packaging RNA (pRNA) three-way junction (3WJ) nanomotifs and seven phi29 pRNA 3WJ deletion mutant nanomotifs were investigated using UV optical melting on a three-component RNA system. Our data reveal that some pRNA 3WJs are more stable than the phi29 pRNA 3WJ. The stability of the 3WJ contributes to the unique self-assembly properties of pRNA. Thus, ultrastable pRNA 3WJ motifs suggest new scaffolds for pRNA-based nanotechnology. We present data demonstrating that pRNA 3WJs differentially respond to the presence of metal ions. A comparison of our data with free energies predicted by currently available RNA secondary structure prediction programs shows that these programs do not accurately predict multibranch loop stabilities. These results will expand the existing parameters used for RNA secondary structure prediction from sequence in order to better inform RNA structure–function hypotheses and guide the rational design of functional RNA supramolecular assemblies.
Highlights
Multibranch loops are key determinants of structural and functional roles in RNA
Thermodynamic parameters determined by UV optical grams used, none accurately predicted either the actual free melting for each construct and its respective 3WJ nanomotif energies of the junctions or variations in phi29 packaging RNA (pRNA)
Among the investigated pRNA 3WJ constructs, the SF5 and M2 pRNA 3WJs were most thermodynamically stable, making them attractive alternatives to the phi29 pRNA 3WJ scaffold used in pRNA-based nanotechnology
Summary
Multibranch loops are key determinants of structural and functional roles in RNA. In prohead RNA (pRNA), an essential component of the phi29-like bacteriophage DNA packaging motor (Guo et al 1987), the three-way junction (3WJ) is a multibranch loop that is a flexible, dynamic region in the RNA (Lescoute and Westhof 2006; Zhang et al 2012, 2013) that helps to correctly place helices in the spatial orientation necessary for packaging (Zhang et al 1997; Hoeprich and Guo 2002; Ding et al 2011; Hill et al 2016). The shape, self-assembly properties, and stability of pRNA can be leveraged in the rational design of functional supramolecular structures, including polyvalent nanoscale delivery systems (Chen et al 2000; Shu et al 2004, 2011b, 2013; Guo 2005; Guo et al 2005, 2006; Li et al 2009, 2015a; Tarapore et al 2011; Haque et al 2012; Khisamutdinov et al 2014). An important consideration in the design of functional nanostructures is the stability of the pRNA “building block” (Shu et al 2011a; Li et al 2015a,b)
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