@book{hardin_ascencio-ibaanez_knopp_2013, title={Experimental biochemistry: Theory, experiment, analysis and reporting}, publisher={Dubuque, Iowa: Kendall Hunt}, author={Hardin, C. and Ascencio-Ibaanez, T. and Knopp, J.}, year={2013} } @article{zhang_foegeding_hardin_2004, title={Effect of sulfated polysaccharides on heat-induced structural changes in beta-lactoglobulin}, volume={52}, ISSN={["1520-5118"]}, DOI={10.1021/jf035037s}, abstractNote={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.}, number={12}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Zhang, GY and Foegeding, EA and Hardin, CC}, year={2004}, month={Jun}, pages={3975–3981} } @article{lieberman_hardin_2004, title={Extraction of information on the buildup and consumption of reactive intermediates from quadruplex DNA assembly time courses}, volume={1679}, ISSN={["0167-4781"]}, DOI={10.1016/j.bbaexp.2004.03.006}, abstractNote={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.}, number={1}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION}, author={Lieberman, DV and Hardin, CC}, year={2004}, month={Jul}, pages={59–64} } @misc{hardin_perry_white_2001, title={Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids}, volume={56}, ISSN={["1097-0282"]}, DOI={10.1002/1097-0282(2000/2001)56:3<147::aid-bip10011>3.0.co;2-n}, abstractNote={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+, NH4+, Sr2+, and Pb2+ 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., Ca2+, Co2+, Mn2+, Zn2+, Ni2+ and Mg2+ 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. © 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 147–194, 2001}, number={3}, journal={BIOPOLYMERS}, author={Hardin, CC and Perry, AG and White, K}, year={2001}, pages={147–194} } @article{ikeda_foegeding_hardin_2000, title={Phospholipid/fatty acid-induced secondary structural change in beta-lactoglobulin during heat-induced gelation}, volume={48}, ISSN={["0021-8561"]}, DOI={10.1021/jf990434h}, abstractNote={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.}, number={3}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Ikeda, S and Foegeding, EA and Hardin, CC}, year={2000}, month={Mar}, pages={605–610} } @misc{hardin_brown_roberts_pelsue_1999, title={Antibodies that selectively bind quadruplex nucleic acids}, volume={6,001,657}, number={1999 Dec. 14}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Hardin, C. C. and Brown, B. A., II and Roberts, J. F. and Pelsue, S. C.}, year={1999} } @article{hardin_sneeden_lemon_brown_guenther_sierzputowska-gracz_1999, title={Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of Hepatitis A virus}, volume={27}, ISSN={["1362-4962"]}, DOI={10.1093/nar/27.2.665}, abstractNote={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.}, number={2}, journal={NUCLEIC ACIDS RESEARCH}, author={Hardin, CC and Sneeden, JL and Lemon, SM and Brown, BA and Guenther, RH and Sierzputowska-Gracz, H}, year={1999}, month={Jan}, pages={665–673} } @article{brown_brown_li_hardin_1998, title={Construction and characterization of a quadruplex DNA selective single-chain autoantibody from a viable motheaten mouse hybridoma with homology to telomeric DNA binding proteins}, volume={37}, ISSN={["0006-2960"]}, DOI={10.1021/bi981434y}, abstractNote={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.}, number={46}, journal={BIOCHEMISTRY}, author={Brown, JC and Brown, BA and Li, YQ and Hardin, CC}, year={1998}, month={Nov}, pages={16338–16348} } @misc{brown_li_brown_hardin_roberts_pelsue_shultz_1998, title={Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice}, volume={37}, ISSN={["0006-2960"]}, DOI={10.1021/bi981354u}, abstractNote={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.}, number={46}, journal={BIOCHEMISTRY}, author={Brown, BA and Li, YQ and Brown, JC and Hardin, CC and Roberts, JF and Pelsue, SC and Shultz, LD}, year={1998}, month={Nov}, pages={16325–16337} } @article{hardin_corregan_lieberman_brown_1997, title={Allosteric interactions between DNA strands and monovalent cations in DNA quadruplex assembly: Thermodynamic evidence for three linked association pathways}, volume={36}, ISSN={["0006-2960"]}, DOI={10.1021/bi970488p}, abstractNote={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)}, number={49}, journal={BIOCHEMISTRY}, author={Hardin, CC and Corregan, MJ and Lieberman, DV and Brown, BA}, year={1997}, month={Dec}, pages={15428–15450} } @misc{myron_williams_hardin_woytek_stephens_1997, title={Occupancy sensor and method of operating same}, volume={5,640,143}, number={1997 Jun. 17}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Myron, D. D. and Williams, E. R. and Hardin, C. C. and Woytek, T. W. and Stephens, M. A.}, year={1997} }