Understanding nucleic acid-ion interactions

Abstract

Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.

Keywords: Hill equation; Manning condensation; Poisson–Boltzmann; RNA/DNA; electrostatics; free energy; ions.

Figure 1

Structures of (a) three typical proteins, (b) the DNA mimetic pentapeptide repeat protein MfpA, and (c) three nucleic acids. Nucleic acids are highly negatively charged compared with proteins. Negatively charged residues are shown in red, positively charged residues in blue, polar residues in green, and nonpolar residues in white. The proteins shown are cytochrome c [Protein Data Bank (PDB) identifier 1CRC (188), 105 residues, and total estimated charge +9e], ribose binding protein [PDB 1URP (189), 271 residues, total charge −2e], and human serum albumin [PDB 1E7H (190), 585 residues, total charge −15e]. (b) The pentapeptide repeat protein MfpA [PDB 2BM4 (191), 186 residues, total charge −5e] inhibits gyrase by binding as a DNA mimetic (191, 192) despite having only a modest overall negative charge. (c) The nucleic acids shown are double-stranded DNA (dsDNA) [PDB 2BNA (193), 24 residues, total charge −22e], double-stranded RNA (dsRNA) [adapted from PDB 3CIY (194), 30 residues, total charge −28e], and the P4–P6 domain from the Tetrahymena group I intron ribozyme [PDB 1GID (21), 158 residues, total charge −157e].

Figure 2

Crystal structures reveal details of ion–nucleic acid interactions but fail to give a complete picture of the ion atmosphere. (a) Crystal structure of the P4–P6 domain of the Tetrahymena group I intron ribozyme [Protein Data Bank (PDB) identifier 1GID] (21). Twelve crystallographically resolved Mg²⁺ ions are shown as blue spheres. (b) Crystal structure of the tandem aptamer of a glycine riboswitch from Fusobacterium nucleatum (PDB 3P49) (195). The two glycine molecules bound to the tandem aptamer are shown in pink. Thirteen crystallographically resolved Mg²⁺ ions are shown as blue spheres. (c) Close-up of the metal ion core of the P4–P6 RNA. Metal ions (spheres) interact with the adenine (A)-rich bulge, making a number of contacts and organizing the RNA residues. (d) Detailed rendering of the glycine binding pocket of aptamer I of the structure in panel b. The two crystallographically resolved Mg²⁺ ions (spheres) coordinate a series of backbone residues in the binding pocket as well as the ligand glycine. These RNAs have 158 and 169 residues, corresponding to total charges of the RNAs of −157 and −168, respectively. The structures resolve 12 and 13 Mg²⁺, respectively, which means that only ~15% of the ion atmosphere required for charge neutrality is accounted for by the explicitly resolved ions. In addition, the crystallographically resolved ions can be replaced by ions of other identities; both RNAs can undergo partial folding in high concentrations of monovalent ions (62, 83). Docking of the P5abc metal ion core in the P4–P6 RNA (c) and glycine binding (d) and folding to the native state for the tandem aptamer riboswitch require divalent ions, however, with limited specificity between different divalent species (52, 62, 103).

Figure 3

Statistical analysis of ions in nucleic acid crystal structures. Distribution of the fraction of nucleic acid charges that are neutralized by ions resolved in the crystal structures of 6,238 nucleic acids obtained from the nucleic acid data base (http://ndbserver.rutgers.edu/). Nucleic acid charges were estimated by counting atoms of type P (phosphorus); ion charges were estimated by counting all elemental ions of the first and second groups as well as Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺. The “fraction neutralized” is defined as the total positive charge due to the ions divided by the total negative charge of the nucleic acid. More than half of all crystal structures of nucleic acids do not resolve any ions, and in the structures that do report ions, the total charge of the crystallographically resolved ions, on average, accounts for only ~20% of the total nucleic acid charge.

Figure 4

Quantifying the total composition of the ion atmosphere. (a) Excess ion density as a function of distance from a nucleic acid, approximated here as a cylinder of radius 10 Å, determined from Poisson–Boltzmann theory. The ion atmosphere does not penetrate into the cylinder; the ion density is highest close to the nucleic acid and equals the bulk concentration far away from the nucleic acid. (b) The excess numbers of ions determined from a molecular dynamics simulation and the total charge of these ions around a 24-bp DNA duplex. The total charge of the ion atmosphere is equal and opposite to the charge of the DNA of −46e (dashed line). The example shown is for 5 mM MgCl₂ and 40 mM NaCl. Data were taken from Reference . (c) Scheme of the buffer equilibration–atomic emission spectroscopy (BE-AES) approach that enables complete quantification of the total ion atmosphere (51, 53). (d) The number of associated excess Na⁺ ions (blue circles), depleted Cl⁻ ions (orange triangles), and their total charge (green squares) around a 24-bp DNA duplex determined by BE-AES. The total charge of the ion atmosphere is equal, to within experimental error, to the inverse of the DNA charge (dashed line). Data are from Reference .

Figure 5

Schematic of the ion atmosphere surrounding nucleic acids under different solution conditions. The circles represent excess counterions and depleted coions in the ion atmosphere, respectively, compared with a buffer-only sample. The number of accumulated and depleted ions shown schematically corresponds (approximately) to the results of buffer equilibration–atomic emission spectroscopy ion counting measurements for 24-bp DNA duplexes (represented by cylinders and molecular renderings on the left of each panel) (51), with each symbol representing two ions. The schematically rendered ion atmospheres depict several experimentally observed trends: (i) Higher total ionic concentration increases coion depletion compared to counterion accumulation, which is partly due to excluded volume effects (see Reference for details); (ii) an atmosphere predominantly of divalent cations leads to a tighter spatial association of the ions around the nucleic acid; (iii) at approximately equal concentrations of mono- and divalent ions, the ion atmosphere is dominated by divalent ions; and (iv) even in a large excess of monovalent ions, some divalent ions are expected to remain close to the nucleic acid.

Figure 6

The interplay of nucleic acid structural ensembles and ion interactions. The tethered duplex system has been used as a model system to study how ion interactions modulate the conformational ensemble of nucleic acids (17, 40, 41). (a,b) Sequence and schematic of the tethered duplex system. The system consists of two 12-bp DNA duplexes (red) joined by a flexible polyethylene glycol tether (green). (c) Visualization of the computationally derived ensemble of the tethered duplex system at various ionic conditions, from left to right and top to bottom: 0.02, 0.06, 0.17, 0.3, 2.0 M monovalent ions; the last image (bottom right) shows the ensemble in the absence of electrostatics (i.e., steric effects only). One duplex is rendered in gray, and the colored balls represent the distal end of the other duplex. Colors represent the energetic difference between the conformer and the minimum-energy conformer observed in the ensemble, from red (<1 k_BT) to blue (>3 k_BT). At a low salt concentration, electrostatic repulsion leads to repulsion between the duplexes; at a higher salt concentration, the repulsion is reduced, and a larger conformational ensemble is explored. Adapted with permission from Reference . Copyright 2008, American Chemical Society.