Richard D. Sheardy

Rational Design of an Imprinted Polymer: Maximizing Selectivity by Optimizing the Monomer–Template Ratio for a Cinchonidine MIP, Prior to Polymerization, Using Microcalorimetry

Abstract Molecular imprinting, a technique that allows for preparation of adsorbents with sites tailored for recognition of a particular molecule, continues to grow because it holds promise for several areas including separations. A key fundamental aspect in forming a molecular imprinted polymer (MIP) is the ability to optimize the amount of functional monomer relative to template used for the polymerization. In this paper, isothermal titration calorimetry (ITC) was used to predict the optimum functional monomer concentration for preparing an MIP for the drug, cinchonidine. Calorimetric titrations of cinchonidine with the functional monomer, methacrylic acid, suggested that a methacrylic acid–cinchonidine hydrogen bonded complex of minimum energy exists for solutions of 4:1 mol/mol of the monomer relative to the template. When this ratio of functional monomer to template was utilized for the MIP synthesis, the resulting polymer displayed significantly better selectivity for cinchonidine than polymers prepared with lesser or greater amounts of methacrylic acid. This observation is explained in terms of a balance that was achieved in the monomer–template equilibrium during the polymerization. This balance maximizes the favorable hydrogen bonding interactions with the template during the polymerization that yields the selective sites, but minimizes the use of excess monomer, which leads to non‐selective sites on the polymer. The results within suggest that ITC can be a valuable tool in the syntheses of MIPs.

Thermodynamics of Forming a Parallel DNA Crossover

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX(1) molecule, wherein one crossover is missing between the two helical domains. The JX(1) molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.

The Facile Synthesis and Characterization of Novel Cationic Metallated and Nonmetallated Tetrapyridino Porphyrazines Having Different Metal Centers

Abstract A porphyrazine and a series of tetracationic porphyrazines, metallated and nonmetallated, were synthesized. The neutral tetrapyridino porphyrazine was obtained in a yield of 45%. The tetracationic nonmetallated tetraethanoltetrapyridino porphyrazinium iodide was obtained in a yield of 79%. The metallated porphyrazines are (Mn+2, Cu+2, Co+2, Zn+2) = M. M‐Tetraethanoltetrapyridino porphyrazinium iodide was obtained in a yield of 78%, 69%, 75%, and 72%, respectively. The porphyrazine and the tetracationic porphyrazines, nonmetallated and metallated, were characterized by elemental analysis, ESI‐MS, MALDI‐MS, FTIR, and UV‐VIS.

Assessing the sequence specificity in the binding of Co(III) to DNA via a thermodynamic approach

The interaction specificities of Co(III) with DNA were investigated via consideration of thermodynamic characteristics of the duplex to single strand transition for DNA oligomers incubated in the presence of [Co(NH3)5(OH2)] (ClO4)3. It has previously been demonstrated that incubation of the DNA oligomer [(5medC‐dG)4]2 with this cobalt complex leads to coordination of the cobalt center to the DNA, presumably at N7 of guanine bases [D. C. Calderone, E. J. Mantilla, M. Hicks, D. H. Huchital, W. R. Murphy, Jr. and R. D. Sheardy, (1995) Biochemistry 34, 13841]. In this report, DNA oligomers of different sequence were incubated with [Co(NH3)5(OH2)] (ClO4)3 via protocols developed previously and the treated oligomers were subjected to thermal denaturation for comparison to the untreated oligomers. The DNA oligomers were designed in order to investigate the sequence specificity, if any, in the reaction of the cobalt complex with DNA. The values of Tm, ΔHuH, and Δn (the differential ion binding term) obtained from the thermal denaturations were used to assess the sequence specificity of the interaction. For all oligomers, treated or untreated, Tm and ΔuH vary linearly with log [Na+] and hence the value of Δn is a function of the Na+ concentration. The results indicate no significant reaction between the cobalt complex and oligomers possessing isolated ‐GA‐ or ‐CG‐ sites; however, the thermodynamic characteristics of DNA oligomers possessing either an isolated ‐GG‐ site or an isolated ‐GC‐ site were altered by the treatment. Atomic absorption studies of the treated oligomers demonstrate that only the DNA oligomers possessing isolated ‐GG‐ or ‐GC‐ sites bind cobalt. Hence, the changes in the thermodynamic properties of these oligomers are a result of cobalt binding with a remarkable sequence specificity.

The interface between an alternating CG motif and a random sequence motif displays altered nuclease activity

Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.

Linking pH, Temperature, and K+ Concentration for DNA i-Motif Formation

The conformation a particular DNA segment assumes depends upon its sequence context and the environment under which it is prepared. To complement our findings with G-rich sequences related to the human telomere, we have been investigating the pH induced transition from single strand to i-motif for sequences related to the human telomere C-rich strand. We have carried out titrations of (CCCTAA)4 from pH 7.0 to pH 5.0 at temperatures ranging from 15 to 45 °C at 115 mM K+ and at K+ concentrations ranging from 15 to 215 mM at 25 °C. Circular dichroism (CD) spectra were determined to monitor the transition. The pH at the midpoint of the proton induced transition, pHmp, is dependent upon both temperature and [K+]. Wyman-type plots of log K vs pH yielded linear correlations and the slopes of those lines, ΔQ, were also linearly dependent on [K+] and T. For these studies, ΔQ represents the minimum number of protons that must be added to the oligomer to induce the initial folding. These results are consistent with Le Chateliers principle. Optical melting studies were also carried out for (CCCTAA)4 at pH 5.0 and [K+] ranging from 15 to 315 mM. Linear correlations between the temperature at the midpoint of the transition, Tm, and log [K+] allowed determination of the differential ion binding term, ΔnK+. These linkages between pH, temperature, and [K+] can be utilized to design i-motif forming DNA oligomers with highly tunable properties.

Stability of the Na+ Form of the Human Telomeric G-Quadruplex: Role of Adenines in Stabilizing G-Quadruplex Structure

G-quadruplexes are higher order DNA structures that play significant roles in gene transcription and telomeric maintenance. The formation and stability of the G-quadruplex structures are under thermodynamic control and may be of biological significance for regulatory function of cellular processes. Here, we report the structural influence and energetic contributions of the adenine bases in the loop sequences that flank G-repeats in human telomeric DNA sequence. Spectroscopic and calorimetric techniques are used to measure the thermal stability and thermodynamic contributions to the stability of human telomeric G-quadruplexes that have been designed with systematic changes of A to T throughout the telomeric sequence. These studies demonstrate that the thermal stability of the G-quadruplex structure is directly related to the number and position of the adenines that are present in the telomeric sequence. The melting temperature (Tm) was reduced from 59 °C for the wild-type sequence to 47 °C for the sequence where all four adenines were replaced with thymines (0123TTT). Furthermore, the enthalpy required for transitioning from the folded to unfolded G-quadruplex structure was reduced by 15 kcal/mol when the adenines were replaced with thymines (37 kcal/mol for the wild-type telomeric sequence reduced to 22 kcal/mol for the sequence where all four adenines were replaced with thymines (0123TTT)). The circular dichroism melting studies for G-quadruplex sequences having a single A to T change showed significantly sloping pretransition baselines and their differential scanning calorimetry (DSC) thermograms revealed biphasic melting profiles. In contrast, the deoxyoligonucleotides having sequences with two or more A to T changes did not exhibit sloping baselines or biphasic DSC thermograms. We attribute the biphasic unfolding profile and reduction in the enthalpy of unfolding to the energetic contributions of adenine hydrogen bonding within the loops as well as the adenine stacking to the G-tetrads of the G-quadruplex structure.

Linking Temperature, Cation Concentration and Water Activity for the B to Z Conformational Transition in DNA

High concentrations of Na+ or [Co(NH3)6]3+ can induce the B to Z conformational transition in alternating (dC-dG) oligo and polynucleotides. The use of short DNA oligomers (dC-dG)4 and (dm5C-dG)4 as models can allow a thermodynamic characterization of the transition. Both form right handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na+ at 25 °C. However, at 2.0 M Na+ or 200 μM [Co(NH3)6]3+, (dm5C-dG)4 assumes a left handed double helical structure (Z-DNA) while the unmethylated (dC-dG)4 analogue remains right handed under those conditions. We have previously demonstrated that the enthalpy of the transition at 25 °C for either inducer can be determined using isothermal titration calorimetry (ITC). Here, ITC is used to investigate the linkages between temperature, water activity and DNA conformation. We found that the determined enthalpy for each titration varied linearly with temperature allowing determination of the heat capacity change (ΔCp) between the initial and final states. As expected, the ΔCp values were dependent upon the cation (i.e., Na+ vs. [Co(NH3)6]3+) as well as the sequence of the DNA oligomer (i.e., methylated vs. unmethylated). Osmotic stress experiments were carried out to determine the gain or loss of water by the oligomer induced by the titration. The results are discussed in terms of solvent accessible surface areas, electrostatic interactions and the role of water.

Novel NMR Techniques to Study Structural and Dynamical Properties of DNA Quadruplexes

Since the right-handed double helical conformation was first described by Watson and Crick (1), we have realized that DNA is highly polymorphic as evidenced by left-handed DNA (2), parallel-stranded DNA (3), DNA quadruplexes (4), and DNA i-motifs (5), to name a few. The conformation a particular segment of DNA assumes, as well as its thermodynamic stability and ligand binding properties, depend highly upon its sequence context and also on environmental conditions (e.g., temperature, cations present, pH, and so on).