An Isothermal Titration and Differential Scanning Calorimetry Study of the G‑Quadruplex DNA–Insulin Interaction

The binding of insulin to the G-quadruplexes formed by the consensus sequence of the insulin-linked polymorphic region (ILPR) was investigated with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The thermal denaturation temperature of insulin was increased by almost 4 °C upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (<i>K</i>_D, Δ<i>H</i>, Δ<i>G</i>, and Δ<i>S</i>) of the insulin–G-quadruplex complex were further investigated by temperature-dependent ITC measurement over the range of 10–37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which is mainly driven by entropic factors below 25 °C but by enthalpic terms above 30 °C. The interaction was also examined in several different buffers, and results showed that the observed Δ<i>H</i> is dependent on the ionization enthalpy of the buffer used. This indicates proton release upon the binding of G-quadruplex DNA to insulin. Additionally, the large negative change in heat capacity for this interaction may be associated with the dominant hydrophobicity of the amino acid sequence of insulin’s β subunit, which is known to bind to the ILPR G-quadruplex DNA

Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-Conjugated Dendrimer to Bacterial Cell-Wall Peptide

Multivalent ligand–receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5­(Van)_<i>n</i> (<i>n</i> = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly­(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5­(Van)₄ bound most tightly with a <i>K</i>_D of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5­(Van)₁ or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5­(Van)_<i>n</i> and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at <i>n</i> = 4 greater than 96 pN at <i>n</i> = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates

Force Spectroscopy of Multivalent Binding of Riboflavin-Conjugated Dendrimers to Riboflavin Binding Protein

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5­(RF)_<i>n</i> nanoparticle. By varying the average RF ligand valency (<i>n</i> = 0, 3, 5), the rupture force was measured between G5­(RF)_<i>n</i> and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (<i>n</i> = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell–Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates

Characterization of Folic Acid and Poly(amidoamine) Dendrimer Interactions with Folate Binding Protein: A Force-Pulling Study

Atomic force microscopy force-pulling experiments have been used to measure the binding forces between folic acid (FA) conjugated poly­(amidoamine) (PAMAM) dendrimers and folate binding protein (FBP). The generation 5 (G5) PAMAM conjugates contained an average of 2.7, 4.7, and 7.2 FA per dendrimer. The most probable rupture force was measured to be 83, 201, and 189 pN for G5-FA_2.7, G5-FA_4.7, and G5-FA_7.2, respectively. Folic acid blocking experiments for G5-FA_7.2 reduced the frequency of successful binding events and increased the magnitude of the average rupture force to 274 pN. The force data are interpreted as arising from a network of van der Waals and electrostatic interactions that form between FBP and G5 PAMAM dendrimer, resulting in a binding strength far greater than that expected for an interaction between FA and FBP alone

Atomic Force Microscopy Probing of Receptor–Nanoparticle Interactions for Riboflavin Receptor Targeted Gold–Dendrimer Nanocomposites

Riboflavin receptors are overexpressed in malignant cells from certain human breast and prostate cancers, and they constitute a group of potential surface markers important for cancer targeted delivery of therapeutic agents and imaging molecules. Here we report on the fabrication and atomic force microscopy (AFM) characterization of a core–shell nanocomposite consisting of a gold nanoparticle (AuNP) coated with riboflavin receptor-targeting poly­(amido amine) dendrimer. We designed this nanocomposite for potential applications such as a cancer targeted imaging material based on its surface plasmon resonance properties conferred by AuNP. We employed AFM as a technique for probing the binding interaction between the nanocomposite and riboflavin binding protein (RfBP) in solution. AFM enabled precise measurement of the AuNP height distribution before (13.5 nm) and after chemisorption of riboflavin-conjugated dendrimer (AuNP–dendrimer; 20.5 nm). Binding of RfBP to the AuNP–dendrimer caused a height increase to 26.7 nm, which decreased to 22.8 nm when coincubated with riboflavin as a competitive ligand, supporting interaction of AuNP–dendrimer and its target protein. In summary, physical determination of size distribution by AFM imaging can serve as a quantitative approach to monitor and characterize the nanoscale interaction between a dendrimer-covered AuNP and target protein molecules in vitro

Biophysical Characterization of a Riboflavin-Conjugated Dendrimer Platform for Targeted Drug Delivery

The present study describes the biophysical characterization of generation-five poly­(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have <i>K</i>_D values of ≥465 nM on a riboflavin basis, an affinity ∼93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein–conjugate stability is achieved with the N-3 series and at lower ligand valency

Self-propagating, protease-resistant, recombinant prion protein conformers with or without <i>in vivo</i> pathogenicity

<div><p>Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert <i>in vivo</i>. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res’ pathogenicity, and revealed that the <i>in vivo</i> seeding ability of rPrP-res does not necessarily result in pathogenicity.</p></div

RT-QuIC detection of seeding activities in rPrP-res^RNA, rPrP-res^RNA-low and brain homogenates from mice inoculated with rPrP-res^RNA or rPrP-res^RNA-low using Bank Vole rPrP.

<p>(<b>A</b>) rPrP-res^RNA (R, upper panel) and rPrP-res^RNA-low (R-low, lower panel) with designated dilutions were used to seed RT-QuIC reactions. (<b>B</b>) Western blots of PK digested RT-QuIC products seeded by rPrP-res^RNA (R) and rPrP-res^RNA-low (R-low) (Left panel) and by brain homogenates from mice 354 (inoculated with rPrP-res^RNA-low) and 361 (inoculated with rPrP-res^RNA) (Right panel). Each lane represents the RT-QuIC product collected from one single well. (<b>C</b>) RT-QuIC reactions were seeded with 10⁻³ brain tissue dilution from rPrP-res^RNA (Br 361, 362 and 365, upper panel) or rPrP-res^RNA-low (Br 352, 353 and 354, lower panel) inoculated mice. R and R-low in the parentheses indicate the inocula. (<b>D</b>) End-point quantitation of prion seeding activity in the original inocula (rPrP-res^RNA and rPrP-res^RNA-low) and the brain homogenates of mice inoculated with rPrP-res^RNA (Br 361, 362 and 365) or rPrP-res^RNA-low (Br 352, 353 and 354). na, not available.</p

Comparisons between rPrP-res^RNA, rPrP-res^RNA-low and rPrP-res^NIH.

<p>(<b>A</b>) rPrP-res^RNA (R), rPrP-res^RNA-low (R-low) and rPrP-res^NIH (NIH) were PK-digested, and PK-resistant PrP fragments were detected by western blotting using POM1 (left panel) and then 3F10 (right panel) anti-PrP monoclonal antibodies. (<b>B</b>) Representative sPMCA reactions seeded by rPrP-res^RNA-low (R-low) or rPrP-res^NIH (NIH). The PK-resistant fragment of rPrP-res^RNA (R) and undigested rPrP (C) were used as controls. (<b>C</b>) Representative sPMCA reactions seeded by rPrP-res^NIH (NIH) with complete (+) or cofactor-free (-) substrates. C, undigested rPrP as controls. (<b>D</b>) GdnHCl denaturation curve for rPrP-res^NIH. Two-way ANOVA analyses followed by Tukey’s multiple comparisons test in GraphPad Prism revealed that there is no significant difference between either rPrP-res^RNA (R) and rPrP-res^NIH (NIH) or rPrP-res^RNA-low (R-low) and rPrP-res^NIH (NIH).</p

The propagation of both pathogenic rPrP-res^RNA and non-pathogenic rPrP-res^RNA-low is cofactor-dependent.

<p>(<b>A</b>) In seeded sPMCA reactions, rPrP-res^RNA (R) or rPrP-res^RNA-low (R-low) were added to complete substrates (+cofactors) and to substrates without any cofactor (-cofactors). The mixtures were subjected to 3 rounds of sPMCA as indicated. After each round, 10 μL of PMCA product was collected and subjected to PK digestion, SDS-PAGE, and western blotting. C: undigested rPrP as controls. (<b>B</b>) Six rounds of sPMCA seeded by rPrP-res^RNA with (+cofactors) or without cofactors (-cofactors). After each round, 10 μL of PMCA product was collected and subjected to PK digestion, SDS-PAGE, and western blotting. C: rPrP as controls. (<b>C</b>) The Elispot cell infection assay of round-6 sPMCA products from panel <b>B</b>.</p