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. 2011 Feb 8;50(5):640-53.
doi: 10.1021/bi101470n. Epub 2011 Jan 14.

Fluorescence competition and optical melting measurements of RNA three-way multibranch loops provide a revised model for thermodynamic parameters

Biao Liu  1 , Joshua M Diamond, David H Mathews, Douglas H Turner
Affiliations
Free PMC article

Fluorescence competition and optical melting measurements of RNA three-way multibranch loops provide a revised model for thermodynamic parameters

Biao Liu et al. Biochemistry. .
Free PMC article

Abstract

Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.

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Figures

Figure 1
Figure 1
(A) Illustration of fluorescence competition assay to measure the free energy of multibranch loop formation. As the competitor strand, C, is titrated into the solution of “reference” structure, RS, the competitor strand will substitute the fluorescein-labeled short strand in the “reference” structure to form the new multibranch loop, MBL. The fluorescein-labeled short strand, FS, is freed from the “reference” structure, and the fluorescence intensity of the solution changes. (B) Typical titration curve and fitting for FCA. Here the system is G_CG_G/Ca3C (Table 1). The “reference” structure concentration is 30 μM before titration, and the competitor strand is 400 μM in the titration solution.
Figure 2
Figure 2
Sequence design. Group 1 is designed to study the influence of the number and type of unpaired nucleotides on multibranch loop stability. (A) “Reference” structure for fluorescence competition assay to analyze the free energy of systems in group 1. (B) Secondary structures of systems studied. Group 2 is designed to investigate the stability of a loop E motif (5′GAA/3′AUGA) in a multibranch loop. (C) “Reference” structure for fluorescence competition assay to analyze the free energy of systems in group 2. The noncanonical base pairing and some tertiary interactions in the loop E motif are shown (78). (D) A variant of loop E motif in multibranch loop.
Figure 3
Figure 3
Optical melting of three-way multibranch loops G_CG_G/CaC at 44.5 μM (black squares), G_CG_G/Ca2C at 25.0 μM (red diamond), and CgaaaCGaG/CcaguaG at 23.5 μM (green triangle).
Figure 4
Figure 4
Diagram of multibranch loop dissociation in melting. As the multibranch loop dissociates, the newly released nucleotides from the left and right helices stack onto the hairpin stem, so that the first mismatch and second and third 3′ dangling nucleotides stabilize the hairpin.
Figure 5
Figure 5
ΔG°37,MBL init plots for G_CG_G/CnmC. Black rectangles are for G_CG_G/CamC measured by UV melting, red circles are for G_CG_G/CumC measured by UV melting, green triangles are for G_CG_G/CamC measured by FCA, blue upside-down triangles are for G_CG_G/CumC measured by FCA, purple left-facing triangles are for CgaaaCGaG/CcaguaG and CgaaaCGaG/CcagaG measured by UV melting, burgundy right-facing triangles are for CgaaaCGaG/CcaguaG and CgaaaCGaG/CcagaG measured by FCA, and cyan diamonds are for G_CG_G/CnmC predicted by eq 24.
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