Search

Understanding the underlying mechanisms of COVID-19 progression and the impact of various pharmaceutical interventions is crucial for the clinical management of the disease. We developed a comprehensive mathematical framework based on the known mechanisms of the SARS-CoV-2 virus infection, incorporating the renin-angiotensin system and ACE2, which the virus exploits for cellular entry, key elements of the innate and adaptive immune responses, the role of inflammatory cytokines and the coagulation cascade for thrombus formation. The model predicts the evolution of viral load, immune cells, cytokines, thrombosis, and oxygen saturation based on patient baseline condition and the presence of co-morbidities. Model predictions were validated with clinical data from healthy people and COVID-19 patients, and the results were used to gain insight into identified risk factors of disease progression including older age, co-morbidities such as obesity, diabetes, and hypertension, and dysregulated immune response 1,2 . We then simulated treatment with various drug classes to identify optimal therapeutic protocols. We found that the outcome of any treatment depends on the sustained response rate of activated CD8 + T cells and sufficient control of the innate immune response. Furthermore, the best treatment -or combination of treatments - depends on the pre-infection health status of the patient. Our mathematical framework provides important insight into SARS-CoV-2 pathogenesis and could be used as the basis for personalized, optimal management of COVID-19.

The emergence and worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused major disruptions to the healthcare system and medical education. In response, the scientific community has been acquiring, releasing, and publishing data at a remarkable pace. At the same time, medical practitioners are taxed with greater professional duties than ever before, making it challenging to stay current with the influx of medical literature., To address the above mismatch between data release and provider capacity and to support our colleagues, physicians at the Massachusetts General Hospital have engaged in an electronic collaborative effort focused on rapid literature appraisal and dissemination regarding SARS-CoV-2 with a focus on critical care., Members of the Division of Pulmonary and Critical Care, the Division of Cardiology, and the Department of Medicine at Massachusetts General Hospital established the Fast Literature Assessment and Review (FLARE) team. This group rapidly compiles, appraises, and synthesizes literature regarding SARS-CoV-2 as it pertains to critical care, relevant clinical questions, and anecdotal reports. Daily, FLARE produces and disseminates highly curated scientific reviews and opinion pieces, which are distributed to readers using an online newsletter platform., Interest in our work has escalated rapidly. FLARE was quickly shared with colleagues outside our division, and, in a short time, our audience has grown to include more than 4,000 readers across the globe., Creating a collaborative group with a variety of expertise represents a feasible and acceptable way of rapidly appraising, synthesizing, and communicating scientific evidence directly to frontline clinicians in this time of great need., (Copyright © 2020 by the American Thoracic Society.)

In this work, we describe a benchtop model that recreates the motion and function of the diaphragm using a combination of advanced robotic and organic tissue. First, we build a high-fidelity anthropomorphic model of the diaphragm using thermoplastic and elastomeric material based on clinical imaging data. We then attach pneumatic artificial muscles to this elastomeric diaphragm, pre-programmed to move in a clinically relevant manner when pressurized. By inserting this diaphragm as the divider between two chambers in a benchtop model-one representing the thorax and the other the abdomen-and subsequently activating the diaphragm, we can recreate the pressure changes that cause lungs to inflate and deflate during regular breathing. Insertion of organic lungs in the thoracic cavity demonstrates this inflation and deflation in response to the pressures generated by our robotic diaphragm. By tailoring the input pressures and timing, we can represent different breathing motions and disease states. We instrument the model with multiple sensors to measure pressures, volumes, and flows and display these data in real-time, allowing the user to vary inputs such as the breathing rate and compliance of various components, and so they can observe and measure the downstream effect of changing these parameters. In this way, the model elucidates fundamental physiological concepts and can demonstrate pathology and the interplay of components of the respiratory system. This model will serve as an innovative and effective pedagogical tool for educating students on respiratory physiology and pathology in a user-controlled, interactive manner. It will also serve as an anatomically and physiologically accurate testbed for devices or pleural sealants that reside in the thoracic cavity, representing a vast improvement over existing models and ultimately reducing the requirement for testing these technologies in animal models. Finally, it will act as an impactful visualization tool for educating and engaging the broader community., (© 2020 Author(s).)

Vascular leakage, protein exudation, and edema formation are events commonly triggered by inflammation and facilitated by gaps that form between adjacent endothelial cells (ECs) of the vasculature. In such paracellular gap formation, the role of EC contraction is widely implicated, and even therapeutically targeted. However, related measurement approaches remain slow, tedious, and complex to perform. Here, we have developed a multiplexed, high-throughput screen to simultaneously quantify paracellular gaps, EC contractile forces, and to visualize F-actin stress fibers, and VE-cadherin. As proof-of-principle, we examined barrier-protective mechanisms of the Rho-associated kinase inhibitor, Y-27632, and the canonical agonist of the Tie2 receptor, Angiopoietin-1 (Angpt-1). Y-27632 reduced EC contraction and actin stress fiber formation, whereas Angpt-1 did not. Yet both agents reduced thrombin-, LPS-, and TNFα-induced paracellular gap formation. This unexpected result suggests that Angpt-1 can achieve barrier defense without reducing EC contraction, a mechanism that has not been previously described. This insight was enabled by the multiplex nature of the force-based platform. The high-throughput format we describe should accelerate both mechanistic studies and the screening of pharmacological modulators of endothelial barrier function.

In endothelial gap formation, local tractions exerted by the cell upon its basal adhesions are thought to exceed balancing tensile stresses exerted across the cell-cell junction, thus causing the junction to rupture. To test this idea, we mapped evolving tractions, intercellular stresses, and corresponding growth of paracellular gaps in response to agonist challenge. Contrary to expectation, we found little to no relationship between local tensile stresses and gap formation. Instead, we discovered that intercellular stresses were aligned into striking multi-cellular domains punctuated by defects in stress alignment. Surprisingly, gaps emerged preferentially not at stress hotspots, as predicted, but rather at stress defects. This unexpected behavior is captured by a minimal model of the cell layer as a jammed assembly of cohesive particles undergoing plastic rearrangements under tension. Together, experiments and model suggest a new physical picture in which gap formation, and its consequent effect on endothelial permeability, is determined not by a local stress imbalance at a cell-cell junction but rather by emergence of non-local, cooperative stress reorganization across the cellular collective., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Endothelial cell alignment along the direction of laminar fluid flow is widely understood to be a defining morphological feature of vascular homeostasis. While the role of associated signaling and structural events have been well studied, associated intercellular stresses under laminar fluid shear have remained ill-defined and the role of these stresses in the alignment process has remained obscure. To fill this gap, we report here the tractions as well as the complete in-plane intercellular stress fields measured within the human umbilical vein endothelial cell (HUVEC) monolayer subjected to a steady laminar fluid shear of 1 Pa. Tractions, intercellular stresses, as well as their time course, heterogeneity, and anisotropy, were measured using monolayer traction microscopy and monolayer stress microscopy. Prior to application of laminar fluid flow, intercellular stresses were largely tensile but fluctuated dramatically in space and in time (317 ± 122 Pa). Within 12 h of the onset of laminar fluid flow, the intercellular stresses decreased substantially but continued to fluctuate dramatically (142 ± 84 Pa). Moreover, tractions and intercellular stresses aligned strongly and promptly (within 1 h) along the direction of fluid flow, whereas the endothelial cell body aligned less strongly and substantially more slowly (12 h). Taken together, these results reveal that steady laminar fluid flow induces prompt reduction in magnitude and alignment of tractions and intercellular stress tensor components followed by the retarded elongation and alignment of the endothelial cell body. Appreciably smaller intercellular stresses supported by cell-cell junctions logically favor smaller incidence of gap formation and thus improved barrier integrity., (Copyright © 2015 the American Physiological Society.)

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.

The principal constituent of the living cell is water. The role of the hydration shell and bulk H(2)O solvent is well recognized in the dynamics of isolated proteins, but the role of water in the dynamics of the integrated living cytoskeleton (CSK) remains obscure. Here we report a direct connection of dynamics of water to dynamics of the integrated CSK. The latter are known to be scale-free and to hinge upon a frequency f(0) that is roughly invariant across cell types. Although f(0) is comparable in magnitude to the rotational relaxation frequency of water (gigahertz range), the physical basis of f(0) remains unknown. Using the human airway smooth muscle cell as a model system, we show here that replacing water acutely with deuterium oxide impacts CSK dynamics in major ways, slowing CSK remodeling dynamics appreciably, and lowering f(0) by up to four orders of magnitude. Although these observations do not distinguish contributions of bulk solvent versus hydration shell, they suggest a unifying hypothesis, namely, that dynamics of integrated CSK networks are slaved in a direct fashion to fluctuations arising in intracellular water.

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell-cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. Within that stress landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behaviour, as do breast cancer cell lines before but not after the epithelial-mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighbouring cells join forces to transmit appreciable normal stress across the cell-cell junction, but migrate along orientations of minimal intercellular shear stress., (© 2011 Macmillan Publishers Limited. All rights reserved)

A method was developed to detect the time course of the overall presence of intermediate species during K+-induced DNA quadruplex assembly from single-stranded d(TG4) oligonucleotides in experiments in which only the combined circular dichroisms (CD) of all species present could be measured directly. The presence of intermediate species is determined unambiguously but quantitative estimates can be made only to the extent that the CD characteristics of all intermediates are known. The method consists of (i) obtaining CD spectra of known concentrations of initial and final species to determine their molar ellipticity coefficients, (ii) carrying out CD measurements of the kinetics of quadruplex assembly reactions at two different wavelengths, chosen to give optimal differentiation between the initial and final species, and (iii) using the results of (ii) to detect discrepancies between the rates of consumption of single strands and the generation of quadruplex to infer the presence of intermediate species. The analysis was facilitated by the validation and use of biphasic exponential expressions obtained from the SAS nonlinear curve fitting procedure NLIN in place of the raw CD data. The general method is described, then applied to data from [d(TG4)4.(K+)3] quadruplex assembly experiments., (Copyright 2004 Elsevier B.V.)

The mechanism that leads to a decreased aggregation of beta-lactoglobulin in the presence of dextran sulfate and lambda-carrageenan was investigated by assessing changes in the denaturation thermodynamics and protein structure. Differential scanning calorimetry results showed that the denaturation temperature (Tp) was about 4.6 degrees C higher in the presence of dextran sulfate, as compared with beta-lactoglobulin alone, whereas in the presence of lambda-carrageenan the difference in Tp was about 1.2 degrees C. Changes in protein structure studies using near-UV circular dichroism (CD) provided support for the calorimetric results. The transition midpoint (Tm) for denaturation of beta-lactoglobulin was about 5 degrees C higher in the presence of dextran sulfate than that found with beta-lactoglobulin alone and about 2 degrees C in the presence of lambda-carrageenan. Thermal modifications of the tertiary structure of beta-lactoglobulin were irreversible at temperatures above 67 degrees C; the addition of dextran sulfate reduced the extent of such modifications. Far-UV CD studies indicated that the addition of dextran sulfate or lambda-carrageenan did not affect secondary structure changes of beta-lactoglobulin upon heating. These studies indicate that dextran sulfate and lambda-carrageenan can enhance the stability of beta-lactoglobulin and thereby inhibit heat denaturation and aggregation.

Effects of phosphatidylcholine (PC) and the predominant fatty acids (FAs) in milk, butyrate, oleate, and palmitate, on secondary structural changes in beta-lactoglobulin (beta-LG) during heat-induced gelation were analyzed on the basis of circular dichroism (CD) spectra. Small-strain oscillatory measurements were carried out to characterize viscoelastic properties of the heat-induced gels. In the absence of added salt, PC and FAs induced helix formation of beta-LG on heating to 80 degrees C and increased the storage moduli (G') of heat-induced gels. In the presence of 500 mM NaCl, PC did not change the CD spectrum of beta-LG but decreased G'. In contrast, butyrate substantially unfolded beta-LG in 500 mM NaCl on heating, forming very elastic gels with increased G' values. Palmitate and oleate induced beta-LG gel formation at 25 degrees C without heating; heating to 80 degrees C almost completely unfolded beta-LG in 500 mM NaCl.

The dissociation and assembly of quadruplex DNA structures (and a few quadruplex RNAs) have been characterized at several levels of rigor, ranging from gross descriptions of factors that govern each process, to semiquantitative comparisons of the relative abilities of these factors to induce stabilization or destabilization, to quantitative studies of binding energies (thermodynamics), transformational rates (kinetics), and analysis of their transition-state energies and mechanisms. This survey classifies these factors, describes the trends and focuses on their interdependencies. Quadruplex assembly is induced most efficiently by added K(+) and elevating the strand concentration; however, Na(+), NH(4)(+), Sr(2+), and Pb(2+) are also very effective stabilizers. Quadruplex dissociation is typically accomplished by thermal denaturation, "melting"; however, when the quadruplex and monovalent cation concentrations are low enough, or the temperature is sufficiently high, several divalent cations, e.g., Ca(2+), Co(2+), Mn(2+), Zn(2+), Ni(2+) and Mg(2+) can induce dissociation. Stabilization also depends on the type of structure adopted by the strand (or strands) in question. Variants include intramolecular, two- and four-stranded quadruplexes. Other important variables include strand sequence, the size of intervening loops and pH, especially when cytosines are present, base methylation, and the replacement of backbone phosphates with phosphorothioates. Competitive equilibria can also modulate the formation of quadruplex DNAs. For example, reactions leading to Watson-Crick (WC) duplex and hairpin DNAs, triplex DNAs, and even other types of quadruplexes can compete with quadruplex association reactions for strands. Others include nonprotein catalysts, small molecules such as aromatic dyes, metalloporphyrins, and carbohydrates (osmolytes). Other nucleic acid strands have been found to drive quadruplex formation. To help reinforce the implications of each piece of information, each functional conclusion drawn from each cited piece of thermodynamic or kinetic data has been summarized briefly in a standardized table entry., (Copyright 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 147-194, 2001)

Two RNA fragments from the region just upstream of the internal ribosome entry site of Hepatitis A virus (HAV) were studied, a 35mer (HAV-35), 5'U4C3U3C3U4C3U3C2UAU2C3U33(4), and a 23mer (HAV-23), 5(4)U4C3U3C3U4C3U33(4). Secondary structural predictions and nuclease digestion patterns obtained with genomic RNAs suggested that they link two stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conventional WC secondary structure, but do fold to form a condensed, stacked 'domain'. To obtain more information, folding of HAV-23 and -35 RNA fragments was characterized using 1H nuclear magnetic resonance, in H2O as a function of pH and temperature, circular dichroism as a function of NaCl concentration, pH and temperature, and square-wave voltammetry as a function of pH. The results indicate that these oligo-nucleotides form intramolecular structures that contain transient U*U base pairs at pH 7 and moderate ionic strength (100 mM NaCl). This folded structure becomes destabilized and loses the U*U base pairs above and below neutral pH, especially at ionic strengths above 0.1. All of the cytidine protons exchange relatively rapidly with solvent protons (exchange lifetimes shorter than 1 ms), so the structure contains few if any C*CH+base pairs at neutral pH, but can apparently form them at pH values below 6. We present a series of possible models in which chain folding draws the strand termini closer together, possibly serving to pull the attached WC hairpin domains together and providing a functional advantage by nucleating reversible formation of a more viable RNA substrate.

An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.

The series of cooperative transitions that lead to [d(TG4)4.(K+)m] quadruplex assembly upon rapid addition of KCl to d(TG4) strands were studied. Quadruplex samples were dialyzed against KCl then Li-EDTA and found to retain between three and five strongly bound potassiums with affinities >10(6) M-2. Absorbance thermal denaturation (melt) and circular dichroism (CD) equilibrium binding data were obtained. The latter were analyzed using two classes of binding models to simulate the effects of the assumed intermolecular interactions on the binding curves (isotherms). The melt experiments yielded equilibrium dissociation constants (Kd) ranging from 10(-11) to 10(-12) M3 at the melting temperatures. Extrapolating these values to 23 degrees C predicts Kd values in the 10(-28) M3 range if the heat capacity (Cp) is not strongly dependent upon temperature changes over this range. Assuming Ka is equal to 1/Kd (from melting analyses), very large association free energies stabilize the quadruplex at 23 degrees C in 100 mM KCl (DeltaGa = -43 kcal mol-1). Plots of the differential melt curve peak half-widths, a measure of cooperativity, versus d(TG4) concentration showed that quadruplex dissociation is much more cooperative at 400 mM KCl than at 100 mM KCl. Forty-eight hour quadruplex assembly time courses were monitored by CD at 264 nm. Equilibrium quadruplex accumulation generally required over 10 h, and net reaction extents were in the 10-85% range. Hill plots of the data show that initial steps in the multistep pathway are positively cooperative, presumably due to strong strand-cation and strand-strand binding interactions in duplex and triplex assembly reactions, then negatively cooperative in quadruplex formation. Models were developed to rationalize the experimental observations in terms of consecutive cooperative allosteric transitions from cation-deficient relaxed (R) strand-aggregates to cation-containing tense (T) structures, driven by the allosteric effector K+. Quantitative mappings of positive and then negative cooperativity were obtained by fitting the results as a function of strand number incorporated during quadruplex assembly. Surprisingly, models for reactions involving incorporation of five and six strands fit the data better than models involving only four strands. The 5-step "induced fit" model fits the data as well as or better than 3- and 4-step models and better than all of the strand aggregation models that were devised and investigated. Net association free energies (summation operatori=1,n) ranged from -20 to -26 kcal mol-1, approximately half the magnitude of the apparent stabilities measured by absorbance melts. Likely explanations for this discrepancy involve hysteresis and errors due to inadequate equilibration in the melt experiments. Hysteresis is thought to be produced by irreversibility due to different predominant mechanisms in absorbance (dissociation) and CD (association) experiments. The kinetic block to quadruplex assembly can be unambiguously attributed to quadruplex formation and not intermediate steps in the assembly mechanism. On the basis of these results we propose that, in addition to the more conventional assembly mechanisms involving duplex dimerization and stepwise strand addition, quadruplex formation can also proceed by triplex-triplex disproportionation. Interaction statistics arguments that support the energetic feasibility of the disproportionation pathway are presented. The allosteric quadruplex assembly model provides a mechanism which could be used by the cell to simultaneously modulate DNA structure and activity within telomeres, transcriptional promoters, recombination-prone chromatin, and other G-rich DNAs. As a result of this allosterism, cation and strand availability and strand-pairing capabilities could profoundly influence the functional capacity of a particular strand over a relatively narrow range of effector concentration changes. (ABSTRACT TRUNCATED)

Initiation of translation of hepatitis A virus (HAV) RNA occurs by internal entry and is likely to involve the interaction of trans-acting cellular protein factors with cis-acting structural elements of an internal ribosomal entry segment (IRES) within the 5'-nontranslated RNA. To characterize interactions between African green monkey kidney (BS-C-1) cell proteins and the predicted stem-loop IIIa (nucleotides 155-235) located at the 5' border of the HAV IRES, we utilized an electrophoresis mobility shift assay (EMSA) to identify a 39-kDa RNA-binding protein (p39). Amino-terminal amino acid sequencing of highly purified p39 revealed absolute identity with human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The identity of p39 as simian GAPDH was further confirmed by antigenic and biochemical similarities between p39 and human GAPDH. Analysis of the RNA binding properties of simian GAPDH revealed that this cellular protein interacts with two additional sites in the HAV 5'-nontranslated RNA, one located between nucleotides 1-148 and the other between nucleotides 597-746. Competitive EMSAs also demonstrated that GAPDH and human polypyrimidine tract-binding protein, a putative picornavirus translation initiation factor, compete with each other for binding to stem-loop IIIa, suggesting that the relative cytoplasmic abundance of GAPDH and polypyrimidine tract-binding protein in individual cell-types may be an important determinant of viral translation activity. Human GAPDH was found to destabilize the folded structure of the stem-loop IIIa RNA based upon observed decreases in the circular dichroism spectra of this RNA following binding of the protein. This RNA helix-destabilizing activity of GAPDH could directly influence IRES-dependent translation and/or replication of picornavirus RNA.

An autoantibody specific for a DNA quadruplex structure has been isolated and cloned from three-month-old autoimmune "viable motheaten" mice. This antibody (mev-alpha Q1) has been tested extensively in vitro and found to bind specifically and preferentially to the parallel-stranded quadruplex structure formed by the oligonucleotide d(CGC G4 GCG). The anti-quadruplex antibody does not show specific affinity for single-, double-, or triple-stranded oligonuclotides of similar CG-rich sequence motifs.

We want to understand how environmental factors influence zymogen activation of the "cloaked' active site of the 'Ribosome Inactivating Protein' (RIP) from corn kernels. In this study, we focus on how likely chemical effectors in the immediate environment of the 'lid' conspire to unleash the active site upon encountering target membranes of invading pests. Octanol-H2O partitioning free energies of peptides which (i) straddle the proteolysis site, and (ii) form the 'side' and 'bottom' of the proposed 'lid' were found to only slightly favor H2O, suggesting that the peptide is poised to detach from the less polar surface surrounding the RIP active site. Circular dichroism results obtained upon catalase/H2O2 oxidation of the 'lid' peptide suggest that the structure shifts from primarily alpha-helical to primarily beta-like. These results suggest that the active site is more easily 'uncloaked' as a result of the lowered solvent polarity conditions and higher oxidant concentrations in the presence of pest membranes encountered during crucial stages of seed germination.

Incubation of beta-lactoglobulin with immobilized trypsin at 5-10 degrees C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR 5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48-101 and 41-100. Prior to reduction, beta-lactoglobulin C-terminal residues 149-162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel beta-sheet structure similar to the native protein but the alpha-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated delta GDH20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display a pH-dependent binding to immobilized trans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9 degrees C as compared with 81.1 degrees C for native protein.

Previous spectroscopic studies demonstrated that the oligodeoxynucleotide d(CGC G3 GCG) undergoes a reversible cation-dependent transition between Watson-Crick (WC) hairpin and parallel-stranded "G-DNA" quadruplex structures [Hardin, C.C., Watson, T., Corregan, M., & Bailey, C. (1992) Biochemistry 31, 833-841]. The relative stabilities of the structures were assessed as a function of pH, and it was found that the quadruplex was substantially stabilized (delta Tm = +15 degrees C) when the pH was shifted from 7.5 to 6 (apparent pKa = 6.8). In the present study, the effects of different cations and pH on four specific sequence varients were determined to test the proposal that this stabilization is due to C.C+ base pair formation mediated by N3-protonation of cytosine. Characteristically large differences in stability were observed when structures formed by d(TAT G3 ATA) and d(TAT G4 ATA) were thermally dissociated at pH 7 in the presence of different cations, verifying that Gn tracts bordered by TAT- and -ATA sequences form quadruplex structures. Imino proton NMR results indicate that the d(m5C G m5C G3 G m5C G)4 and d(TAT G4 ATA)4 quadruplex structures are parallel-stranded. It was necessary to increase the K+ concentration from 40 mM to ca. 200 mM to stabilize d(TAT G3 ATA)4, while the d(TAT G4 ATA)4 complex was nearly as stable as the quadruplex formed by d(CGC G3 GCG) under the same conditions. The d(TAT G4 ATA)4 quadruplex was only slightly stabilized at pH 6 relative to pH 7.5 (delta Tm = +3 degrees C), confirming that the unique stabilization that occurs in the pH 6.8 range with [d(CGC Gn GCG)4.ionn] complexes is due to the C residues. The sequence d(m5C G m5C G3 G m5C G) was found to form a very stable quadruplex in K+ or Ca2+. As with the quadruplex formed by the unmethylated analog, the stability is greatly enhanced when the pH is decreased below about 7.2 (pKa,obs = 6.8). Dissociation kinetic constants and activation energies were determined for quadruplexes formed by d(CGC G3 GCG), d(m5C G m5C G3 G m5C G) and d(TAT G4 ATA). Quantitative comparisons showed that methylation produces a complex that is much more stable at pH 7 in 40 mM Na+ than either of the unmodified structures; the rate-limiting activation energy for dissociation of d(CGC G3 GCG)4 was 22 kcal mol-1 less than for the methylated analog.(ABSTRACT TRUNCATED AT 400 WORDS)

In 10 mM sodium phosphate, pH 7.6, containing 0.1 mM ethylenediaminetetraacetic acid, ions correspondings to the non-calent, four-stranded oligonucleotide, d(CGCG4GCG)4, were detected by negative ion electrospray ionization (ESI) mass spectrometry at a low nozzle-skimmer (delta NS) bias (-150 V), but not at a higher delta NS bias (> -250 V). In contrast, when the sample was desalted and analyzed by ESI mass spectrometry at a low delta NS bias only ions for the single-stranded d(CGCG4GCG) species were observed. These data agree with spectroscopic evidence which showed that oligonucleotides with the sequence motif 5'd(CGCGnGCG)3', where n = 2-5, formed stable four-stranded complexes in the presence of monatomic cations, like K+, Ca2+, Na+ and Li+, but not in their absence.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH-beta chain amino acid sequences 33-53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990), Biochemistry 29, 1194-1200], 81-95 [Santa-Coloma, T. A., Reichert, L. E., Jr. (1990), J. Biol. Chem. 265, 5037-5042], and the combined sequence (33-53)-(81-95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991), Mol. Cell. Endocrinol. 78, 197-204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH-beta-(33-53), H2N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel beta-pleated sheet, turns including a beta-turn, "other" structures, and a small amount of alpha-helix. The major characteristics of the structure were found to be relatively stable at acidic pH and the predominant effect of increased solvent polarity was a small increase in alpha-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution at pH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH-beta-(33-53). These features included an antiparallel beta-sheet (residues 38-51 and 46-48), turns within residues 41-46, and 50-52 (a beta-turn) and a small N-terminal helical region comprised of amino acids 34-36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to the trans conformation, whereas proline-42 favored the trans conformer (approximately 70%) over the cis (approximately 30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containing trans-42 and trans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure.(ABSTRACT TRUNCATED AT 400 WORDS)

The DNA oligonucleotide d(CGCG3GCG) can form either a Watson-Crick (WC) hairpin or a parallel-stranded quadruplex structure containing six G-quartet base pair assemblies. The exchange between these forms and single strands can be monitored using circular dichroism (CD). NMR results verified the assignment of specific CD bands to quadruplex and hairpin species, respectively. Cations stabilize the quadruplex in the order K+ greater than Ca2+ greater than Na+ greater than Mg2+ greater than Li+ and K+ greater than Rb+ greater than Cs+, indicating that K+ has an optimum ionic radius for complex formation and that ionic charge affects the extent of ion-induced stabilization. The quadruplex is stable in the presence of 40 mM K+ at micromolar DNA concentration and can be kinetically trapped as a metastable form when prepared at millimolar DNA concentration and then diluted into buffer containing 40 mM Na+. The concentration of K+ required to reverse the equilibrium from the hairpin to the quadruplex decreases sharply with increased DNA concentration. The quadruplex has an unusual pKa of ca. 6.8, indicating that C.C+ base pairs are probably forming. This system provides insights into some of the detailed structural characteristics of a ["G4-DNA".ion] complex and an experimental model for the recently proposed "sodium-potassium conformational switch" [Sen, D., & Gilbert, W. (1988) Nature 334, 364-366; Sen, D., & Gilbert, W. (1990) Nature 344, 410-414]. These results may help to explain the lack of cytidine residues in G-rich telomeric DNAs and suggest that methylation of GC-rich duplex DNAs in "GpC islands" may induce quadruplex formation within heterochromatin domains, resulting in reversible chromosomal condensation.

Telomeric DNA consists of G- and C-rich strands that are always polarized such that the G-rich strand extends past the 3' end of the duplex to form a 12-16-base overhang. These overhanging strands can self-associate in vitro to form intramolecular structures that have several unusual physical properties and at least one common feature, the presence of non-Watson-Crick G.G base pairs. The term "G-DNA" was coined for this class of structures (Cech, 1988). On the basis of gel electrophoresis, imino proton NMR, and circular dichroism (CD) results, we find that changing the counterions from sodium to potassium (in 20 mM phosphate buffers) specifically induces conformational transitions in the G-rich telomeric DNA from Tetrahymena, d(T2G4)4 (TET4), which results in a change from the intramolecular species to an apparent multistranded structure, accompanied by an increase in the melting temperature of the base pairs of greater than 25 degrees, as monitored by loss of the imino proton NMR signals. NMR semiselective spin-lattice relaxation rate measurements and HPLC size-exclusion chromatography studies show that in 20 mM potassium phosphate (pH 7) buffer (KP) TET4 is approximately twice the length of the form obtained in 20 mM sodium phosphate (pH 7) buffer (NaP) and that mixtures of Na+ and K+ produce mixtures of the two forms whose populations depend on the ratio of the cations. Since K+ and NH4+ are known to stabilize a parallel-stranded quadruplex structure of poly[r(I)4], we infer that the multistranded structure is a quadruplex. Our results indicate that specific differences in ionic interactions can result in a switch in telomeric DNAs between intramolecular hairpin-like or quadruplex-containing species and intermolecular quadruplex structures, all of which involve G.G base pairing interactions. We propose a model in which duplex or hairpin forms of G-DNA are folding intermediates in the formation of either 1-, 2-, or 4-stranded quadruplex structures. In this model monovalent cations stabilize the duplex and quadruplex forms via two distinct mechanisms, counterion condensation and octahedral coordination to the carbonyl groups in stacked planar guanine "quartet" base assemblies. Substituting one of the guanosine residues in each of the repeats of the Tetrahymena sequence to give the human telomeric DNA, d(T2AG3)4, results in less effective K(+)-dependent stabilization. Thus, the ion-dependent stabilization is attenuated by altering the sequence. Upon addition of the Watson-Crick (WC) complementary strand, only the Na(+)-stabilized structure dissociates quickly to form a WC double helix.(ABSTRACT TRUNCATED AT 400 WORDS)

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions.

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA1Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The 19F resonances serve as sensitive monitors of tRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range. All have pKa's close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNAVal1, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H2O to 2H2O. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M. (1977) J. Biol. Chem. 252, 4418-4420] that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems.(ABSTRACT TRUNCATED AT 400 WORDS)

The equilibrium binding of the ethidium cation (Etd+) to the right-handed A-form of poly-[r(C-G)], the B-form of poly[d(C-G)], and the left-handed Z-forms of Br-poly[r(C-G)] and Br-poly[d(C-G)] was investigated in 0.22 M NaCl by optical methods. Scatchard analysis indicates that Etd+ intercalates into right-handed forms of poly[r(C-G)] and poly[d(C-G)] in a noncooperative manner. Correlation of Etd+ absorbance binding isotherms and polynucleotide circular dichroism data indicates that drug binding to Br-poly[r(C-G) and Br-poly[d(C-G)] results in cooperative conversion from left-handed Z-forms to right-handed intercalated conformations. Approximate stoichiometries necessary to induce the left- to right-handed transitions are 1 Etd+/9 base pairs (bp) for Z-RNA and 1 Etd+/6 bp for Z-DNA. The apparent limiting binding stoichiometries are approximately 1 Etd+/3 bp for RNA and 1 Etd+/2 bp for DNA. The equilibrium binding constants for binding to the right-handed forms decrease in the order Br-poly[d(C-G)], Br-poly[r(C-G)], poly[d(C-G)], and poly[r(C-G)]. Thermodynamic parameters are obtained by van't Hoff analysis of Etd+ absorbance thermal dissociation data. Enthalpy values for all four polynucleotides are negative and of similar magnitude. Negative entropy values indicate that the binding processes are primarily enthalpically driven.(ABSTRACT TRUNCATED AT 250 WORDS)

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.

Features of the 19F nuclear magnetic resonance (NMR) spectra of three purified 5-fluorouracil-(FUra-) substituted Escherichia coli tRNAs, tRNA(1Val), tRNA(mMet), and tRNA(fMet), are compared. Each of the tRNA species can be resolved into two isoaccepting forms, A and B, whose 19F NMR spectra differ in the shift of one peak from the 4.5 to 4.8 parts per million (ppm) range (FUra = O) in the spectrum of isoacceptor B upfield to ca. -15 ppm in that of isoacceptor A. Because the sequences of the two isoacceptors of each tRNA differ only at one position in the D loop, that normally occupied by a dihydrouridine residue, we assign the 4.5 ppm peak in the spectrum of fluorine-labeled tRNA(1Val) to FUra17 and the resonance at 4.6 ppm in the spectrum of fluorouracil-substituted tRNA(mMet) to FUra20. A reciprocal 19F[19F] nuclear Overhauser effect is observed between the downfield peaks A and B in the 19F NMR spectrum of 19F-labeled tRNA(1Val). Assuming that fluorine-labeled tRNA(1Val) has a structure similar to that of yeast tRNA(Phe), only FUra54 and -55 are close enough (4-5 A) to give an appreciable 19F homonuclear Overhauser effect. Peaks A and B have therefore been assigned to FUra54 and -55. As the temperature is raised from 30 to 45 degrees C, the intensity of peak B (6.6 ppm) in the spectrum of 19F-labeled tRNA(1Val) gradually shifts upfield to 6.4 ppm (Tm = 36 degrees C), indicating a temperature-dependent slow exchange of the corresponding 5-fluorouracil residue between two magnetically distinct environments.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR and circular dichroism studies of the duplex formed by the self-complementary DNA hexanucleotide d(C3G3) indicate that it is a B-type structure but differs from standard B-form. An analysis of NMR coupling constants within the deoxyribose moieties yields a 70% or greater contribution from pseudorotation phase angles corresponding to the C3'-exo conformation, a conformation similar to the C2'-endo conformation associated with B-form DNA. Intranucleotide interproton distances are consistent with a B-form structure, but some internucleotide distances are intermediate between A- and B-form structures. Circular dichroism spectra have B-form characteristics but also include an unusual negative band at 282 nm. The solution spectroscopic results are in contrast with X-ray crystallographic studies which find A-form structures for similar sequences.

Chemically brominated poly[r(C-G)] [Br-poly[r(C-G)]] containing 32% br8G and 26% br5C was recently shown to contain a 1:1 mixture of A- and Z-form unmodified nucleotides under physiological conditions of temperature, pH, and ionic strength [Hardin, C. C., Zarling, D. A., Puglisi, J. D., Trulson, M. O., Davis, P. W., & Tinoco, I., Jr. (1987) Biochemistry 26, 5191-5199]. Proton NMR results show that more extensive bromination of poly[r(C-G)] (49% br8G, 43% br5C) produces polynucleotides containing greater than 80% unmodified Z-form nucleotides. Using these polynucleotides as antigens, polyclonal antibodies were elicited in rabbits and mice specific for the Z-form of RNA. IgG fractions were purified from rabbit anti-Br-poly[r(C-G)] sera and characterized by immunoprecipitation, nitrocellulose filter binding, and ELISA. Two different anti-Z-RNA IgG specificities were observed. Decreased levels of brominated nucleotides in the immunogen correlated with an increased extent of specific cross-reactivity with Z-DNA. Inoculation of rabbits with polynucleotide immunogens containing 49% br8G and 43% of br5C produced specific anti-Z-RNA IgGs that do not recognize Z-DNA determinants. This suggests that the 2'-OH group is part of the anti-Z-RNA IgG determinant. In contrast, Br-poly[r(C-G)] immunogens containing 32% br8G and 26% br5C produced IgGs that specifically recognize both Z-RNA and Z-DNA. These results show that the bromine atoms are not required for recognition of the Z conformation by the antibodies. The affinity of these anti-Z-RNA IgGs for Z-RNA is about 10-fold higher than for Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNAMetf has been further characterized. This substituent is responsible for the 19F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution 19F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield 19F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNAMetm and tRNAVal1, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with 19F spectrum of the model compound 5'-deoxy-5-fluoro-5,6-dihydrouridine (dH56FUrd) (delta FUra = -31.4 ppm; JHF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH56FUrd is considerably more alkali labile than 5,6-dihydrouridine (H56Urd). At pH 8.9, where H56Urd is stable, dH56FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a 19F resonance at - 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The 19F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the 19F signal at - 15 ppm. Hydrolysis of this tRNA with RNAase T2 produces a sharp doublet 33 ppm upfield (JHF = 45 Hz). Similarities of the 19F chemical shift and coupling constant to those of dH56FUrd, allows assignment of the peak at -33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.