·C Wobble Formation in Double-Stranded DNA.
Journal of the American Chemical Society
2017; 139 (22): 7540-7548
The high charge density of nucleic acids and resulting ion atmosphere profoundly influence the conformational landscape of RNA and DNA and their association with small molecules and proteins. Electrostatic theories have been applied to quantitatively model the electrostatic potential surrounding nucleic acids and the effects of the surrounding ion atmosphere, but experimental measures of the potential and tests of these models have often been complicated by conformational changes and multisite binding equilibria, among other factors. We sought a simple system to further test the basic predictions from electrostatics theory and to measure the energetic consequences of the nucleic acid electrostatic field. We turned to a DNA system developed by Bevilacqua and co-workers that involves a proton as a ligand whose binding is accompanied by formation of an internal AH(+)·C wobble pair [Siegfried, N. A., et al. Biochemistry, 2010, 49, 3225]. Consistent with predictions from polyelectrolyte models, we observed logarithmic dependences of proton affinity versus salt concentration of -0.96 ± 0.03 and -0.52 ± 0.01 with monovalent and divalent cations, respectively, and these results help clarify prior results that appeared to conflict with these fundamental models. Strikingly, quantitation of the ion atmosphere content indicates that divalent cations are preferentially lost over monovalent cations upon A·C protonation, providing experimental indication of the preferential localization of more highly charged cations to the inner shell of the ion atmosphere. The internal AH(+)·C wobble system further allowed us to parse energetic contributions and extract estimates for the electrostatic potential at the position of protonation. The results give a potential near the DNA surface at 20 mM Mg(2+) that is much less substantial than at 20 mM K(+) (-120 mV vs -210 mV). These values and difference are similar to predictions from theory, and the potential is substantially reduced at higher salt, also as predicted; however, even at 1 M K(+) the potential remains substantial, counter to common assumptions. The A·C protonation module allows extraction of new properties of the ion atmosphere and provides an electrostatic meter that will allow local electrostatic potential and energetics to be measured within nucleic acids and their complexes with proteins.
View details for DOI 10.1021/jacs.7b01830
View details for PubMedID 28489947
Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway
Journal of the American Chemical Society
Decades of study of the RNA folding problem have revealed that diverse and complex structured RNAs are built from a common set of recurring structural motifs, leading to the perspective that a generalizable model of RNA folding may be developed from understanding of the folding properties of individual structural motifs. We used single-molecule fluorescence to dissect the kinetic and thermodynamic properties of a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor (TL/TLR). Our results revealed a multistep TL/TLR folding pathway in which preorganization of the ubiquitous AA-platform submotif precedes the formation of the docking transition state and tertiary A-minor hydrogen bond interactions form after the docking transition state. Differences in ion dependences between TL/TLR variants indicated the occurrence of sequence-dependent conformational rearrangements prior to and after the formation of the docking transition state. Nevertheless, varying the junction connecting the TL/TLR produced a common kinetic and ionic effect for all variants, suggesting that the global conformational search and compaction electrostatics are energetically independent from the formation of the tertiary motif contacts. We also found that in vitro-selected variants, despite their similar stability at high Mg2+ concentrations, are considerably less stable than natural variants under near-physiological ionic conditions, and the occurrence of the TL/TLR sequence variants in Nature correlates with their thermodynamic stability in isolation. Overall, our findings are consistent with modular but complex energetic properties of RNA structural motifs and will aid in the eventual quantitative description of RNA folding from its secondary and tertiary structural elements.
View details for DOI 10.1021/jacs.7b08870
View details for PubMedCentralID PMC5748328
- Electric Field Modulation of Silicon upon Tethering of Highly Charged Nucleic Acids. Capacitive Studies on DNA-modified Silicon (111) ELECTROANALYSIS 2016; 28 (10): 2367-2372
Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?
Journal of the American Chemical Society
2016; 138 (34): 10925-10934
Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other molecules, and the energetics of these processes are profoundly influenced by the ion atmosphere that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atmosphere, understanding its properties and effects will require synergy between computational modeling and experiment. Prior computational models and experiments suggest that cation occupancy in the ion atmosphere depends on the size of the cation. However, the computational models have not been independently tested, and the experimentally observed effects were small. Here, we evaluate a computational model of ion size effects by experimentally testing a blind prediction made from that model, and we present additional experimental results that extend our understanding of the ion atmosphere. Giambasu et al. developed and implemented a three-dimensional reference interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helices, and this model predicts that Na(+) would outcompete Cs(+) by 1.8-2.1-fold; i.e., with Cs(+) in 2-fold excess of Na(+) the ion atmosphere would contain an equal number of each cation (Nucleic Acids Res. 2015, 43, 8405). However, our ion counting experiments indicate that there is no significant preference for Na(+) over Cs(+). There is an ∼25% preferential occupancy of Li(+) over larger cations in the ion atmosphere but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, we followed the folding of the P4-P6 RNA and showed that differences in folding with different alkali metal ions observed at high concentration arise from cation-anion interactions and not cation size effects. Overall, our results provide a critical test of a computational prediction, fundamental information about ion atmosphere properties, and parameters that will aid in the development of next-generation nucleic acid computational models.
View details for DOI 10.1021/jacs.6b04289
View details for PubMedID 27479701
View details for PubMedCentralID PMC5010015
- DNA Intercalators for Detection of DNA Hybridisation: SCS(MI)-MP2 Calculations and Electrochemical Impedance Spectroscopy CHEMPLUSCHEM 2016; 81 (7): 604-612
- Potential-Assisted DNA Immobilization as a Prerequisite for Fast and Controlled Formation of DNA Monolayers ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 2015; 54 (50): 15064-15068
- Cation-Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting. Journal of the American Chemical Society 2015; 137 (46): 14705-14715
Competitive interaction of monovalent cations with DNA from 3D-RISM.
Nucleic acids research
2015; 43 (17): 8405-8415
The composition of the ion atmosphere surrounding nucleic acids affects their folding, condensation and binding to other molecules. It is thus of fundamental importance to gain predictive insight into the formation of the ion atmosphere and thermodynamic consequences when varying ionic conditions. An early step toward this goal is to benchmark computational models against quantitative experimental measurements. Herein, we test the ability of the three dimensional reference interaction site model (3D-RISM) to reproduce preferential interaction parameters determined from ion counting (IC) experiments for mixed alkali chlorides and dsDNA. Calculations agree well with experiment with slight deviations for salt concentrations >200 mM and capture the observed trend where the extent of cation accumulation around the DNA varies inversely with its ionic size. Ion distributions indicate that the smaller, more competitive cations accumulate to a greater extent near the phosphoryl groups, penetrating deeper into the grooves. In accord with experiment, calculated IC profiles do not vary with sequence, although the predicted ion distributions in the grooves are sequence and ion size dependent. Calculations on other nucleic acid conformations predict that the variation in linear charge density has a minor effect on the extent of cation competition.
View details for DOI 10.1093/nar/gkv830
View details for PubMedID 26304542
- Intercalation of Proflavine in ssDNA Aptamers: Effect on Binding of the Specific Target Chloramphenicol ELECTROANALYSIS 2015; 27 (8): 1836-1841
Electrical Potential-Assisted DNA Hybridization. How to Mitigate Electrostatics for Surface DNA Hybridization
ACS APPLIED MATERIALS & INTERFACES
2014; 6 (24): 21851-21858
Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.
View details for DOI 10.1021/am5027902
View details for Web of Science ID 000347139400012
View details for PubMedID 25102381
- The Effect of Interfacial Design on the Electrochemical Detection of DNA and MicroRNA Using Methylene Blue at Low-Density DNA Films CHEMELECTROCHEM 2014; 1 (1): 165-171
Kinetic and thermodynamic hysteresis imposed by intercalation of proflavine in ferrocene-modified double-stranded DNA.
2013; 14 (10): 2208-2216
Surface-confined immobilized redox species often do not show the expected zero peak separation in slow-scan cyclic voltammograms. This phenomenon is frequently associated to experimental drawbacks and hence neglected. However, a nonzero peak separation, which is common to many electrochemical systems with high structural flexibility, can be rationally assigned to a thermodynamic hysteresis. To study this phenomenon, a surface-confined redox species was used. Specifically, a DNA strand which is tagged with ferrocene (Fc) moieties at its 5' end and its complementary capture probe is thiolated at the 3' end was self-assembled in a monolayer at a Au electrode with the Fc moieties being located at the bottom plane of the double-stranded DNA (dsDNA). The DNA-bound Fc undergoes rapid electron transfer with the electrode surface as evaluated by fast scan cyclic voltammetry. The electron transfer is sensitive to the ion transport along the DNA strands, a phenomenon which is modulated upon specific intercalation of proflavine into surface-bound dsDNA. The electron transfer rate of the Fc(0/+) redox process is influenced by the cationic permselectivity of the DNA monolayer. In addition to the kinetic hindrance, a thermodynamic effect correlated with changes in the activity coefficients of the Fc(0/+) moieties near the gold-dsDNA interface is observed and discussed as source of the observed hysteresis causing the non-zero peak separation in the voltammograms.
View details for DOI 10.1002/cphc.201300045
View details for PubMedID 23674389