Dustin B. McIntosh

Sequence-Dependent Elasticity and Electrostatics of Single-Stranded DNA: Signatures of Base-Stacking

Base-stacking is a key factor in the energetics that determines nucleic acid structure. We measure the tensile response of single-stranded DNA as a function of sequence and monovalent salt concentration to examine the effects of base-stacking on the mechanical and thermodynamic properties of single-stranded DNA. By comparing the elastic response of highly stacked poly(dA) and that of a polypyrimidine sequence with minimal stacking, we find that base-stacking in poly(dA) significantly enhances the polymers rigidity. The unstacking transition of poly(dA) at high force reveals that the intrinsic electrostatic tension on the molecule varies significantly more weakly on salt concentration than mean-field predictions. Further, we provide a model-independent estimate of the free energy difference between stacked poly(dA) and unstacked polypyrimidine, finding it to be ∼-0.25 kBT/base and nearly constant over three orders of magnitude in salt concentration.

Single-stranded nucleic acid elasticity arises from internal electrostatic tension

Significance Charged, flexible polymers, such as single-stranded nucleic acids (ssNAs), are ubiquitous in biology and technology. Quantitative description of their solution conformation has remained elusive due to the competing effects of polymer configurational freedom and salt-screened electrostatic repulsion between monomers. We investigate this by measuring the elastic response of single ssNA molecules over a range of salt concentrations. The data are well described by a model, inspired by a mean-field approach, in which intrapolymer electrostatic repulsion creates a salt-dependent internal tension whose interplay with the external force determines the elasticity. The internal tension can be related to the polymer’s charge spacing; thus, our results show how mesoscopic polymer conformation emerges from microscopic structure. Understanding of the conformational ensemble of flexible polyelectrolytes, such as single-stranded nucleic acids (ssNAs), is complicated by the interplay of chain backbone entropy and salt-dependent electrostatic repulsions. Molecular elasticity measurements are sensitive probes of the statistical conformation of polymers and have elucidated ssNA conformation at low force, where electrostatic repulsion leads to a strong excluded volume effect, and at high force, where details of the backbone structure become important. Here, we report measurements of ssDNA and ssRNA elasticity in the intermediate-force regime, corresponding to 5- to 100-pN forces and 50–85% extension. These data are explained by a modified wormlike chain model incorporating an internal electrostatic tension. Fits to the elastic data show that the internal tension decreases with salt, from >5 pN under 5 mM ionic strength to near zero at 1 M. This decrease is quantitatively described by an analytical model of electrostatic screening that ascribes to the polymer an effective charge density that is independent of force and salt. Our results thus connect microscopic chain physics to elasticity and structure at intermediate scales and provide a framework for understanding flexible polyelectrolyte elasticity across a broad range of relative extensions.

Ionic excesses and entropies in mean-field screening models

We examine how the mean-field ionic screening excess associated with a charged macromolecule depends upon the macromolecules geometric conformation and net charge. Our approach involves expanding about Debye–Huckel theory, which allows us to derive a series of general results that establish explicit connections between the ionic excess, non-linear screening mechanisms, and the mobile ion entropy. We conclude with an analysis of the ionic screening entropy in familiar models for polyelectrolyte force-extension behavior, and we find the changes in this entropy are often quite substantial relative to the total free energy differences characterizing a polyelectrolytes extended and compact states.

Electrostatic Effects of Multivalent Salts on SsDNA Elasticity

Nucleic acids are highly-charged polyelectrolytes whose structure and function strongly depend on the concentration and type of salt ions in solution. We have created a simple experimental system for studying interactions between nucleic acids and salt ions, based on magnetic-tweezer measurements of the elasticity of single denatured ssDNA molecules in solutions with a known salt concentration. Using this system, we were able to reconcile single-molecule force-extension data with scaling theories of self-avoiding polymers, and we found that the Kuhn length of ssDNA scales with the Debye length in NaCl solutions (Saleh et al., PRL 102, 068301 (2009)). Here, we use the system to investigate interactions of ssDNA with multivalent salts. We find that, in divalent salt, ssDNA elasticity is qualitatively similar to that in monovalent salt, but with significant quantitative differences. Notably, at low ionic strength, ssDNA in divalent salt maintains the same low-force scaling behavior (‘Pincus blob’ regime) as seen in monovalent salts. However, there are differences in the elastic behavior at high forces (> a few pN). In addition, analysis of the low-force scaling behavior indicates it requires ∼100 fold smaller concentrations of divalent salt to condense ssDNA. We discuss the data in the context of electrostatic theories, including Debye-Huckel, as well as bulk experiments on similar systems.

Elastic Behavior of ssDNA in Salty Solutions

The interaction between highly charged poly-ions, such as DNA, and the smaller ions in salty solutions is of fundamental importance to the basic processes of molecular biology (e.g., ion-mediated nucleic acid folding, collapse and stabilization of proteins). Despite its importance, this phenomenon is poorly understood, particularly for multivalent ions where mean-field theories (e.g. Debye-Huckel) break down. By stretching single denatured ssDNAs in monovalent salt solutions, we have established that force-extension measurements directly and quantitatively probe electrostatic effects on charged polymers in solution (O.A. Saleh et al., PRL 102, 068301 (2009)). We exploited access to the ‘tensile blob’ regime to show that, for a broad range of NaCl concentrations, ssDNA behaves as a real polymer in good solvent with a Kuhn length linearly proportional to the Debye length. Here, we present data on the effects of cations with different valences and chemistries on ssDNA structure. We find that the effects of divalent ions greatly exceed those predicted by simple Debye-Huckel calculations and discuss our data in the context of more realistic theories.