Ionic strength-dependent persistence lengths of single-stranded RNA and DNA

Abstract

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (l(p)) of ssRNA. We observe valence and ionic strength-dependent differences in l(p) in a direct comparison between 40-mers of deoxythymidylate (dT(40)) and uridylate (rU(40)) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.

Fig. 1.

The FRET-averaged end-to-end distance, was calculated from measured values of the Förster radius R₀ and the average FRET efficiencies E_FRET for dT₄₀ (filled) and rU₄₀ (unfilled) in NaCl (Left) and MgCl₂ (Right). Error bars are standard errors from combined uncertainties in R₀ and E_FRET. The decrease in end-to-end distance is expected as excess cations screen the negatively charged nucleic acid backbone, resulting in greater chain flexibility at high salt. While rU₄₀ and dT₄₀ are the same within error up to approximately 200 mM NaCl, they diverge at high salt, with dT₄₀ appearing more compact. Similar trends are observed in Mg (Right).

Fig. 2.

The presence of an attached duplex changes the measured flexibility of ssDNA. The 40-mer construct with (dT₄₀-duplex, triangles) and without (dT₄₀, circles) an attached 18-bp duplex DNA exhibit different smFRET-averaged end-to-end distances, , at all NaCl concentrations measured. This indicates that the duplex excludes the single-stranded chain from its vicinity and may explain the discrepancy between our smFRET measurements of freely diffusing dT₄₀ and previous studies using tethered DNA that report more extended chains.

Fig. 3.

(A) SAXS curves for 75 μM dT₄₀ (circles) and rU₄₀ (triangles) in 100 mM NaCl are shown together with a WLC model for the scattering (solid lines) that optimizes the fit to SAXS data and smFRET data ( for both rU and dT). Experimental uncertainties were used as weights in the fit. The best-fit values of L and l_p predict values of 68.0 Å for dT and 67.2 Å for rU. The WLC scattering curves include an overall scale factor and an additional free parameter, R_cs, to correct for finite molecular cross-section (see Materials and Methods) equal to 4.2 Å for rU and 3.7 Å for dT. In the inset (B), the persistence length l_p and the contour length L of each WLC are shown as symbols with ellipses that enclose the 95% confidence interval. The nonoverlapping ellipses show that dT₄₀ and rU₄₀ are described by WLCs with significantly different properties: dT₄₀ has a longer L and a shorter l_p than rU₄₀. The cartoon depictions of ssDNA (C) and ssRNA (D) illustrate this difference: the RNA chain is drawn with a shorter distance between monomers and a less flexible backbone.

Fig. 4.

The ionic strength dependence of the persistence length (l_p) for dT₄₀ (Left) and rU₄₀ (Right) is different in the presence of monovalent NaCl (filled symbols) and divalent MgCl₂ (unfilled symbols). The x-axis is the activity corrected ionic strength and is represented on a logarithmic scale. Solid lines indicate fits to a phenomenological model for the ionic strength dependence (see Eq. 1). For dT₄₀: b_Na⁺ = 5.83 ± 0.53 M^-1, b_Mg²⁺ = 168 ± 23 M^-1, l_p(∞) = 7.5 ± 0.5 Å, l_p(0) = 20.9 ± 0.5 Å . For rU₄₀: b_Na⁺ = 6.86 ± 0.71 M^-1, b_Mg²⁺ = 264 ± 30 M^-1, l_p(∞) = 10.1 ± 0.6 Å, l_p(0) = 25.2 ± 0.4 Å. The scaling exponent, n, is shown in the figure.