Nichola C. Garbett

Circular dichroism to determine binding mode and affinity of ligand–DNA interactions

Circular dichroism (CD) is a useful technique for an assessment of DNA-binding mode, being a more accessible, low-resolution complement to NMR and X-ray diffraction methods. Ligand–DNA interactions can be studied by virtue of the interpretation of induced ligand CD signals resulting from the coupling of electric transition moments of the ligand and DNA bases within the asymmetric DNA environment. This protocol outlines methods to determine the binding mode and affinity of ligand–DNA interactions and takes approximately 7.5 h.

Differential scanning calorimetry of blood plasma for clinical diagnosis and monitoring.

Differential scanning calorimetry (DSC) provides a useful method to study the unfractionated plasma proteome. Plasma from healthy individuals yields a reproducible signature thermogram which results from the weighted sum of the thermal denaturation of the most abundant plasma proteins. Further investigation of the thermogram for healthy individuals showed it to be sensitive to ethnicity and gender. DSC analysis of plasma from diseased individuals revealed significant changes in the thermogram which are suggested to result not from changes in the concentration of the major plasma proteins but from interactions of small molecules or peptides with these proteins. Closer examination of the diseased thermograms showed a thermogram characteristic of each disease. For cervical cancer, the DSC method yields a progressively shifted thermogram as the disease advances from pre-invasive conditions to late stage cancer. Our application of the DSC method has provided a potential tool for the early diagnosis, monitoring and screening of cancer patients.

Thermodynamic Studies for Drug Design and Screening

Introduction: A key part of drug design and development is the optimization of molecular interactions between an engineered drug candidate and its binding target. Thermodynamic characterization provides information about the balance of energetic forces driving binding interactions and is essential for understanding and optimizing molecular interactions. Areas covered: This review discusses the information that can be obtained from thermodynamic measurements and how this can be applied to the drug development process. Current approaches for the measurement and optimization of thermodynamic parameters are presented, specifically higher throughput and calorimetric methods. Relevant literature for this review was identified in part by bibliographic searches for the period 2004 – 2011 using the Science Citation Index and PUBMED and the keywords listed below. Expert opinion: The most effective drug design and development platform comes from an integrated process utilizing all available information from structural, thermodynamic and biological studies. Continuing evolution in our understanding of the energetic basis of molecular interactions and advances in thermodynamic methods for widespread application are essential to realize the goal of thermodynamically driven drug design. Comprehensive thermodynamic evaluation is vital early in the drug development process to speed drug development toward an optimal energetic interaction profile while retaining good pharmacological properties. Practical thermodynamic approaches, such as enthalpic optimization, thermodynamic optimization plots and the enthalpic efficiency index, have now matured to provide proven utility in the design process. Improved throughput in calorimetric methods remains essential for even greater integration of thermodynamics into drug design.

Effect of O6-Methylguanine on the Stability of G-Quadruplex DNA

The effects of substitution of O6-methylguanine on the structure and stability of a human telomere quadruplex was studied by circular dichroism, thermal denaturation, analytical ultracentrifugation, and molecular dynamics simulations. The results show that, while quadruplex structures can form containing the modified base, they are much less stable than the normal unmodified structure. The extent of destabilization is critically dependent on the exact position of the modified base within the quadruplex structure.

An integrated molecular dynamics (MD) and experimental study of higher order human telomeric quadruplexes

Structural knowledge of telomeric DNA is critical for understanding telomere biology and for the utilization of telomeric DNA as a therapeutic target. Very little is known about the structure of long human DNA sequences that may form more than one quadruplex unit. Here, we report a combination of molecular dynamics simulations and experimental biophysical studies to explore the structural and dynamic properties of the human telomeric sequence (TTAGGG)(8)TT that folds into two contiguous quadruplexes. Five higher order quadruplex models were built combining known single human telomeric quadruplex structures as unique building blocks. The biophysical properties of this sequence in K(+) solution were experimentally investigated by means of analytical ultracentrifugation and UV spectroscopy. Additionally, the environments of loop adenines were probed by fluorescence studies using systematic single-substitutions of 2-aminopurine for the adenine bases. The comparison of the experimentally determined properties with the corresponding quantities predicted from the models allowed us to test the validity of each of the structural models. One model emerged whose properties are most consistent with the predictions, and which therefore is the most probable structure in solution. This structure features contiguous quadruplex units in an alternating hybrid-1-hybrid-2 conformation with a highly ordered interface composed of loop residues from both quadruplexes.

Detection of Cervical Cancer Biomarker Patterns in Blood Plasma and Urine by Differential Scanning Calorimetry and Mass Spectrometry

Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer health.

Clinical application of plasma thermograms. Utility, practical approaches and considerations

Differential scanning calorimetry (DSC) studies of blood plasma are part of an emerging area of the clinical application of DSC to biofluid analysis. DSC analysis of plasma from healthy individuals and patients with various diseases has revealed changes in the thermal profiles of the major plasma proteins associated with the clinical status of the patient. The sensitivity of DSC to the concentration of proteins, their interactions with other proteins or ligands, or their covalent modification underlies the potential utility of DSC analysis. A growing body of literature has demonstrated the versatility and performance of clinical DSC analysis across a range of biofluids and in a number of disease settings. The principles, practice and challenges of DSC analysis of plasma are described in this article.

Binding: a polemic and rough guide.

Binding is at the center of all of biology with cellular events being mediated by a huge array of highly orchestrated, coupled binding interactions. In order to approach a detailed understanding of the molecular forces that drive these interactions, it is essential to obtain thermodynamic information. A huge body of work already exists that describes the concepts and mathematical tools that are at the foundation of current thermodynamic techniques. The purpose of this chapter is to aid the reader in understanding how to apply the available technologies in extracting meaningful thermodynamic information for binding systems.

Tumor targeted mesoporous silica-coated gold nanorods facilitate detection of pancreatic tumors using Multispectral optoacoustic tomography

Multispectral optoacoustic tomography (MSOT) is an emerging imaging technology that offers several advantages over traditional modalities, particularly in its ability to resolve optical contrast at depth on the microscopic scale. While potential applications include the early detection of tumors below clinical thresholds set by current technology, the lack of tumor-specific contrast agents limits the use of MSOT imaging. Therefore, we constructed highly stable nano-contrast agents by coating gold nanorods (GNRs) with either polyacrylic acid (PAA) or aminefunctionalized mesoporous silica (MS). Syndecan-1, which has been shown to target insulin-like growth factor 1 receptor (IGF1-R) (upregulated in pancreatic tumors), was conjugated on the surface of PAA-coated GNRs (PAA-GNRs) or MS-coated GNRs (MS-GNRs) to create tumor-targeted nanoparticles. In vitro, tumor targeting of nanoparticles was assessed with flow cytometry. In S2VP10L cells (positive for IGF1-R), the syndecan-1 MS-GNRs (Syndecan-MS-GNRs) demonstrated an increase in OA signal, 10x, compared to syndecan-1 PAAGNRs (Syndecan-PAA-GNRs). Minimal binding was observed in MiaPaca-2 cells (negative for IGF1-R). In vivo, tumor specific targeting of Syndecan-MS-GNRs was evaluated using a murine orthotopic pancreatic cancer model. The Syndecan- MS-GNRs demonstrated significantly greater accumulation within pancreatic tumors than in off-target organs such as the liver. Mice implanted with the IGF1-R negative MiaPaca-2 cells did not demonstrate specific tumor targeting. In summary, we report that targeted nano-contrast agents (Syndecan-MS-GNRs) can successfully detect orthotopic pancreatic tumors with minimum off-target binding in vivo using MSOT.

Calorimetric analysis of the plasma proteome: identification of type 1 diabetes patients with early renal function decline.

BACKGROUND Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome. METHODS DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n=15) and matched control subjects (n=14). RESULTS The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status. CONCLUSIONS Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D. GENERAL SIGNIFICANCE DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.