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Molecular switches, with target protein-binding activity controlled by prior binding to specific input stimuli, are ubiquitously used in Nature. However, the emulation of such responsive systems, especially in a de novo fashion, remains a significant challenge. Herein, we disclose a strategy that harnesses an intramolecular β-CD/adamantane host-guest interaction to generate a stabilized DNA hairpin (ΔT(m) = 17 °C) that undergoes an input oligonucleotide (ODN)-selective structural transformation from a stem-loop conformation to a duplex. This ODN-induced conformational switch allows for the transition from an inactive state (wherein the adamantane protein-binding headgroup is encapsulated) to an activated protein-binding complex, with a freely accessible adamantane moiety. Given that hairpin domains can be readily modulated to be responsive to alternative ODN triggering sequences and that encapsulating macrocycles, such as β-CD, are good hosts for a number of protein-binding small molecules, this strategy may furnish a general method to develop ODN-responsive protein-binders.

When he was a postdoc in geochemistry at Caltech, Charles David Keeling found himself ideally prepared for the moment when funding for the International Geophysical Year enabled him to design and build a CO(2) monitoring station on Mauna Loa in Hawaii in 1957. He applied rigorous analytical procedures to a geophysical study with enormous implications for humanity.

In this communication, we disclose a generalizable strategy for developing agents with regulable protein-binding ability. In particular, a responsive DNA-small molecule chimera (DC) 1 consisting of two synthetic protein-binding arms and a core oligonucleotide (ODN) domain is discussed. DC 1 can be cycled from a bidentate intramolecular quadruplex form to a monodentate duplex structure, via addition of external ODN stimuli. Importantly, these distinct secondary structures of 1 lead to significantly different protein-binding abilities, with the bidentate conformation showing a 20-fold enhancement (with a 0.8 microM dissociation constant, Kd) in trypsin-binding potency.

Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.

The deposition of Fe(II) into ferritin using 18O2 as the oxidant was investigated. Only 3-4% of the oxygen atoms in the FeOOH core of ferritin were derived from the oxidant. This was true whether large (1200-1900 Fe/molecule) or small (220-240 Fe/molecule) amounts of iron were added to apoferritin or when iron was added to ferritin already containing 1000 Fe/molecule. Experiments using H218O in the solvent showed that nearly all of the oxygen atoms in the core were derived from solvent. The stoichiometry of the reaction was close to 2 Fe(II)/O2, instead of the expected value of 4 Fe(II)/O2. Reactions of 18O2 with Fe(II) in the absence of apoferritin had a similar 18O distribution and stoichiometry. Our results are most consistent with the crystal growth model of ferritin iron deposition.

The transferrin molecule has two specific metal-binding sites, each of which may provide iron for the biosynthesis of hemoglobin by reticulocytes. Diferric human transferrin was shown to be a better iron donor, per iron atom, for rabbit reticulocytes, than was monoferric transferrin obtained by isoelectric focusing. The difference in binding of 125I-labeled monoferric and differic transferrin to reticulocytes may be sufficient to account for the difference in iron uptake. In contrast, diferric and monoferric rabbit transferrin both donated iron to reticulocytes at the same rate, per iron atom. In an experiment using 55Fe/59Fe doubly labeled transferrin, one iron binding site of human transferrin was a better iron donor than the other. In rabbit transferrin, the two sites appeared to function equivalently. Care was taken in these experiments to demonstrate that labeled iron added to dilute solutions of transferrin was indeed specifically bound to the protein. A liquid scintillation counting procedure, simpler than existing methods, was developed to quantitate 55Fe and 59Fe in blood.

The transfer of iron between horse spleen [55Fe]ferritin and human apotransferrin or [59Fe]transferrin in homogeneous solution was investigated. Transfer between the two proteins in the presence of citrate, ATP, or ascorbate occurs in both direction, but the net flow is always from ferritin to transferrin. Ferritin which is ca. 1/3 to 1/2 saturated with iron appears to be most reactive. Chemically prepared apoferritin does not accept iron from diferric transferrin. Citrate-mediated transfer of iron from ferritin to apotransferrin is first order with respect to ferritin, zero order with respect to transferrin, and has a complex dependence upon citrate concentration. Direct transfer of iron from native or reconstituted ferritin to apotransferrin in the absence of any identifiable mediating agent was observed to occur at about half the rate attained in the presence of 1 mM citrate. No transfer of iron between the two proteins occurs across a dialysis membrane in the absence of a mediating agent. No binding of transferrin and ferritin to each other was demonstrable. One possible explanation for these observations is that iron from the core of ferritin is in equilibrium with iron near the outer surface of the protein, where the metal would be available to transferrin.