The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemi-protonation of cytosine results in a stable C-C+ base pair. Fundamental studies to understand iM formation from C-rich strands of DNA are described. We present a systematic characterization of the consequences of epigenetic modifications, molecular crowding, degree of hydration, and DNA sequence on the stabilities of iM-forming sequences. We used a number of biophysical techniques to characterize both the folded iM and the folding kinetics of an iM. We established a mechanism for the folding. We observed that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also observed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding as compared to those bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded form. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and can be used to determine the pKa of an iM. Our results indicate that 5-hydroxymethylation of cytosine destabilized the iMs against thermal and pH-dependent melting, while 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions, the thermal stability of iMs increased and the pKa was raised to near 7.0. Taken together, our work has laid the foundation for examining folding and structural changes in more complex iMs.

Block ionomer complex (BIC) dissociation and the subsequent effects on gene "knockdown" are reported. Aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization was utilized to prepare a statistical macro chain transfer agent (macroCTA) consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA); HPMA confers water stability, and APMA provides a facile pathway for the conjugation of folic acid, a cellular targeting moiety. This macroCTA was chain extended with N,N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), thus preparing hydrophilic-block-cationic copolymers with varying repeat units of DMAPMA. After end-group removal and folic acid conjugation, these hydrophilic-block-cationic copolymers were complexed with small interfering RNA (siRNA) and GLuc DNA, a double stranded DNA (dsDNA) analogue of siRNA. These complexes were prepared at a nitrogen-to-phosphate ratio (N : P) = 1, and complexes prepared with siRNA and GLuc DNA were demonstrated to be comparable; the hydrodynamic radii (Rh) and changes to the secondary structure were identical, while zeta-potential and gel electrophoresis confirmed complex neutrality. Analytical ultracentrifugation (AUC) was utilized to ascertain binding constants and stoichiometry. Increased DMAPMA block length (cationic length) caused an increase in the binding constant, but the stoichiometry remained constant at 1 : 1. Solution differential scanning calorimetry (DSC) was conducted to investigate the BIC stability. Similar to AUC, the melting temperature (Tm) increased with increasing cationic block length, and overall, a shift in Tm of similar to 40 degrees C was observed, indicating that increasing the DMAPMA length confers greater BIC stability. Furthermore, complex dissociation was not observed. Gene down-regulation was monitored in KB cells expressing Gaussia Luciferase, and the time for maximum gene knockdown to occur increased with increasing DMAPMA block length. Given the large binding constants and this increased stability, it can be concluded that in vitro complex dissociation occurs via an ion exchange mechanism.

The development of the fluorescence detection system (Aviv-FDS) for the AUC allows a single fluorescently labeled species to be quantitatively characterized against a highly concentrated and heterogeneous background. During our use of the FDS to characterize ELP, a novel drug delivery vector (see Lyons, et. al., Biophys. J. 104, 2009-2021, 2013), in serum, we encountered the Johnston-Ogston (J-O) effect. The J-O effect is a classical anomaly in sedimentation theory describing the non-ideal sedimentation properties of a component as a function of high concentrations of other components. We examined the J-O effect using recent advances in AUC hardware, the AU-FDS (AVIV Biomedical), and data-analysis methods, primarily Sedanal global direct boundary fitting. We empirically quantified the self and cross-sedimentation non-ideality properties of ELP and the two most ubiquitous serum proteins, Albumin (∼35 mg/ml) and γ-Globulins (∼10-15 mg/ml). We have verified and measured the presence of cross-term hydrodynamic and thermodynamic non-ideality by running SV studies on a fluorescently labeled component (∼100 nM) in a titration experiment with high concentrations of unlabeled components. This has been accounted for through the introduction of a 3x3 non-ideality matrix of Ks and BM1 values into Sedanal. ELP experiments with mixtures of Albumin and γ-Globulins were also performed in an attempt to recapitulate the J-O behavior of a serum solution. Clearly other components or effects contribute to the J-O effect and additional experiments with lipids and PEG solutions are planned. These studies lay the groundwork for bringing quantitative hydrodynamic analyses into crowded environments, and will allow measurement of hydrodynamic and equilibrium macromolecular properties in a physiological state. (Supported by the UMC AUC Facility and NSF MRI grant 1040372.)

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT‐binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p‐binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer–dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function. © 2013 Wiley Periodicals, Inc

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

Elastin-like Polypeptides (ELPs) are genetically engineered biopolymers that are derived from the endogenous protein Tropoelastin. ELPs are structurally disordered and soluble at low temperatures but transition to a β-spiral and aggregate at a Transition Temperature (TT). This aggregation is being explored as a novel drug delivery vector by thermally targeting systemically delivered ELP-drug conjugates. We are investigating the biophysical properties of ELP[V5G3A2-150] as a means of understanding and predicting the behavior in vivo. We have investigated the hydrodynamic, structural and thermodynamic properties of ELP[V5G3A2-150] through the use of CD, turbidity, DLS, DSC and SV. DLS and SV analyses suggest that ELP[V5G3A2-150] experiences small amounts of weak association below the TT that increase with temperature. CD analyses further indicate that below the TT ELP[V5G3A2-150] consists of both disordered (≈75%) and β-conformation (≈25%) and as the temperature and concentration is increased the % β-conformation increases. The temperature & concentration dependence of β-conformation suggests that the weak association can be attributed to heterogeneous β-sheets. SV revealed that above the TT ELP[V5G3A2-150] exhibits a temperature dependent critical concentration (CC). This CC is consistent with the TT and suggests that the TT may be described as a solubility constant. Assembly in serum raises the TT by ≈ 2.5°C. This is opposite to the expected effect of macromolecular crowding and suggests that certain serum proteins may be associating with ELP[V5G3A2-150]. Investigation of this effect through SV was greatly complicated by the presence of the Johnston-Ogston (J-O) effect. Further investigation suggested additional complexity in systems exhibiting the J-O effect than previously reported. Two of the additional complexities already determined are cross-term hydrodynamic non-ideality and high-concentration convection. Additional research into the effects of attaching CPPs to ELP[V5G3A2-150] will be presented. Work supported by NSF ARRA 0959211 grant.

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease (AD). Among the different forms of Aβ aggregates, low molecular weight species ranging between 2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12–18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we present the physiochemical aspects of LFAO-monomer interactions and its implications to AD. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. This unique self-replicating property of LFAOs has opened doors towards mechanisms that may be of profound significance for Alzheimer disease pathology.

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.

Aggregates of amyloid-beta (A beta) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of A beta aggregates, low molecular weight species ranging between similar to 2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such " off-pathway" 12-18-mer species of A beta 42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit A beta 42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Perhaps the largest impediment in cancer therapies has been the inability to effectively target systemically delivered therapeutics to cancerous cells. A recent and novel approach to this problem has been to chemically conjugate the therapeutic to an Elastin-Like-Polypeptide (ELP). ELP is a naturally occurring biomolecule which undergoes structural transitions and aggregates in response to temperatures above its transition temperature (Tt). This allows for the specific targeting of a systemically delivered ELP-drug conjugate to an artificially heated tumor. Turbidity experiments on a specific ELP construct, ELP1 (ELP[V5G3A2-150]), show a gradual increase in turbidity as early as 28°C that progressively increases until approximately 37°C, above which there is a cooperative, concentration dependent increase in turbidity corresponding to aggregation of ELP1. This aggregation is reversible and exhibits hysteresis in response to lower temperatures. Sedimentation Velocity (SV) and Circular Dichroism (CD) experiments show that at 20°C ELP1 adopts a random coil configuration with a S20,W of 1.92 and a f/f0 of 2.57 at c=0. As the temperature is raised to 35°C ELP1 shows small amounts of association and sediments with a S20,W of 2.13 and a f/f0 of 2.32 at c=0. At temperatures greater than the Tt a Critical Concentration (Cc) is observed; at c ≥ Cc ELP1 structurally transitions to a β-spiral and aggregates, and at c < Cc ELP1 sediments at a monomeric S20,W. The Cc is temperature dependent and decreases as the temperature is raised (38°C - 3.55mg/ml, 39°C - 2.22mg/ml & 40°C - 1.89mg/ml, respectively). This data suggests that at low temperatures ELP1 adopts a random coil conformation and that as the Tt is approached ELP1 association and β-spiral conformation become thermodynamically favorable. Future work is planned examining how drug conjugation affects the system and the behavior of ELP1 in plasma using fluorescence equipped AUC.

Targeting splicing machinery components is an underdeveloped strategy for cancer therapy. Uridine-rich small nuclear ribonucleoproteins (UsnRNPs) are essential spliceosome components that recognize splice sites in newly transcribed RNA. The major spliceosomal snRNPs are comprised of UsnRNA bound by a ring of Sm proteins. The survival of motor neuron (SMN) complex provides specificity for binding of Sm proteins to UsnRNAs. Three of the seven proteins that comprise the Sm core possess post-translationally modified C-terminal symmetric dimethylarginine (sDMA) residues which promote binding of these proteins to SMN. Here we describe a peptide inhibitor of sDMA that is capable of interfering with SMN/SmB interaction. The inhibitory peptide was attached to elastin-like polypeptide, a thermally responsive macromolecular carrier, in order to increase its stability and allow enhancement of its cellular uptake by thermal targeting. The fusion polypeptide inhibited the interaction of SMN/SmB, inhibited proliferation, and induced apoptosis in HeLa cells.