Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution

Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na ؉ ), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2-5 kT͞bp. Addition of high concentrations of Na ؉ (1.2 M), Mg 2؉ (0.6 M), and spermidine 3؉ (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated. correlation ͉ DNA ͉ RNA ͉ folding ͉ electrostatics N ucleic acids play a central role in the storage, transmission, and control of genetic information. DNA and RNA associate with numerous protein and nucleic acid partners to form the complexes that participate in and regulate cellular metabolism and form catalytic components of biological machines such as the ribosome. A comprehensive understanding of these biological processes requires understanding the fundamental physical and chemical principles underlying the behaviors of the participating molecules. Nucleic acids are highly charged biopolymers, with one formal negative charge per monomeric unit. There are many examples of specific interactions of nucleic acids with ions in nucleic acidprotein complexes and in folded RNAs (1, 2). Nevertheless, the vast majority of cations associated with a nucleic acid are nonspecifically bound. These counterions condense onto DNA or RNA and form a thermally fluctuating sheath commonly referred to as the ion atmosphere (3, 4). Indeed, order-of-magnitude calculations estimate enormous repulsive energies for RNA in the absence of counterions (e.g., Ϸ10 3 kT for a folded RNA with Ϸ400 nucleotides, the size of the Tetrahymena group I intron). ʈ This repulsive energy is predominantly overcome by electrostatic interactions with the ion atmosphere (3-5). Despite the clear importance of the ion atmosphere, experimental investigation has been difficult. The dynamic nature of the ion atmosphere renders it invisible in crystallographic structures. Progress has been made in the direct ''visualization'' of ion distributions around DNA duplexes in recent neutron scattering (6) and anomalous small angle x-ray scattering (SAXS) experiments that provide constraints on the shape of the ion atmosphere (7). Nevertheless, the energetic effects of this atmosphere still remain unclear. The widely used nonlinear Poisson Boltzmann (NLPB) model can estimate the electrostatic energies for nucleic acid conformations in different ionic condition (8-12). However, this model is incomplete. It does not account for the finite sizes of ions and, of particular importance to this work, positional correlations between discrete ions beyond a mean-field approximation (13). Recent theoretical work indicates that such ion-ion correlation effects can lead to a net attraction for strongly charged polyelectrolytes in the presence of multivalent cations (13-16). This effect can be thought of as analogous to attractive London dispersion forces caused by correlations of electron clouds between two nearby molecules, with the counterions arranged to minimize repulsion between one another and to maximize attractive interactions with both polyelectrolytes. Indeed, DNA condensation has been observed in numerous studies (refs. 17 and 18 and references therein), and a counterion-correlation-attractive force is a leading candidate for explaining this phenomenon (18). More recently, Murthy and Rose (15) have proposed that a counterion-induced attractive force is responsible for parallel helical arrangements common in nucleic acid crystal structures. Furthermore, a rapid ''collapse'' early in Mg 2ϩ -promoted RNA folding has been suggested to arise from analogous attractive forces induced by Mg 2ϩ ions Precise and extensive osmotic stress measurements by Parsegian and colleagues (21-26) have yielded quantitative descriptions of screened electrostatic repulsion, hydration forces, and long-range attractive forces in dense, parallel arrays of DNA double helices with several types of counterions. However, many-body effects (24) and disrupted water structure in these highly dehydrated arrays hinder the direct application of these stress measurements to isolated nucleic acids in solution. Here, we have used a simple tethered DNA in which two DNA duplexes of defined length are connected by a neutral flexible linker (Figs. 1A and 2) to isolate the counterion-modulated interactions between two helices. The results provide estimates for repulsive forces in the presence of low concentrations of monovalent cations and an upper limit for any attractive force in the presence of multivalent cations. Materials and Methods Construction of 12-bp Tethered DNAs and 24-bp Duplex DNA. The 12-bp tethered DNAs (12 /PEG9/ 12 and12 /C3/ 12) and 24-bp duplex were assembled from chemically synthesized oligonucleotides (Qiagen, Valencia, CA, and Integrated DNA Technologies, Coralville, IA) as shown in Abbreviations: SAXS, small angle x-ray scattering; NLPB, nonlinear Poisson Boltzmann. ¶ To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. ʈ The Michel-Westhof model of the native Tetrahymena ribozyme (48) and a model of unfolded ribozyme with helices maximally extended from each other were used to estimate the Coulombic repulsion energy in the absence of counterions. The energies of all pairs of phosphates were calculated according to Coulomb's law and summed to yield the total Coulomb energy of the native, compact molecule relative to the unfolded one

The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs

Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution

Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na(+)), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2–5 kT/bp. Addition of high concentrations of Na(+) (1.2 M), Mg(2+) (0.6 M), and spermidine(3+) (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated

Stimulation of Insulin Fibrillation by Urea-induced Intermediates

Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 m urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased α-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation

Partially Folded Intermediates in Insulin Fibrillation

Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn·HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn·HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn·HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn·HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn·HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to \u3e6 M Gdn·HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn·HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn·HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate

Elucidation of The Molecular Mechanism During The Early Events in Immunoglobulin Light Chain Amyloid Fibrillation: EVIDENCE FOR AN OFF-PATHWAY OLIGOMER AT ACIDIC Ph

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate