Alexander M. Chagovetz

Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins

Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG) and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemimethylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the K(D) for 1xMBD-GFP ranged from 106 to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The K(D) values for nonsymmetrical methylation motifs were consistently greater (>2 muM), but the binding selectivity between symmetric and hemimethylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density.

Real-time DNA microarrays: reality check

DNA microarrays are plagued with inconsistent quantifications and false-positive results. Using established mechanisms of surface reactions, we argue that these problems are inherent to the current technology. In particular, the problem of multiplex non-equilibrium reactions cannot be resolved within the framework of the existing paradigm. We discuss the advantages and limitations of changing the paradigm to real-time data acquisition similar to real-time PCR methodology. Our analysis suggests that the fundamental problem of multiplex reactions is not resolved by the real-time approach itself. However, by introducing new detection chemistries and analysis approaches, it is possible to extract target-specific quantitative information from real-time microarray data. The possible scope of applications for real-time microarrays is discussed.

Preliminary use of differential scanning calorimetry of cerebrospinal fluid for the diagnosis of glioblastoma multiforme

Thermal stability signatures of complex molecule interaction in biological fluids can be measured using a new approach called differential scanning calorimetry (DSC). The thermal stability of plasma proteome has been described previously as a method of producing a disease-specific “signature,” termed thermogram, in several neoplastic and autoimmune diseases. We describe the preliminary use of DSC performed on cerebrospinal fluid (CSF) as a diagnostic tool for the identification of patients with glioblastoma multiforme (GBM). Samples of CSF from nine patients with confirmed GBM were evaluated using DSC, and the thermogram signatures evaluated. These thermograms were compared with thermograms of CSF taken from patients with non-neoplastic conditions such as head trauma, hydrocephalus, or CSF leak. Further analysis was also performed on CSF from patients who had non-GBM neoplastic conditions such as carcinomatosis meningitis or central nervous system lymphoma or leukemia. The DSC thermograms of CSF of the patients with GBM were significantly different when compared with other neoplastic and non-neoplastic cases. The melting temperature of the major transition was shifted by 5°C, which makes it easily distinguishable from control cases. Our results are very preliminary, but it appears that the DSC of CSF has potential utility in diagnostics and monitoring disease progression in GBM patients.

Effects of fill fraction on the capture efficiency of nanoscale molecular transducers.

We study the effects of nanotransducer fill fraction on the molecular capture efficiency contribution to overall sensor response. Contrary to expectation, we show that the relative capture efficiency can exceed the fill fraction in the transient regime. However, at thermodynamic equilibrium, the relative efficiency equals the fill fraction in the probe-limited case. When the number of target molecules in solution is the limiting factor in capture, then gains in capture efficiency can be obtained even at thermodynamic equilibrium. The important implication of these results is that significant intrinsic nanotransducer enhancement is necessary in order to provide net gain in sensor response, but that nanotransducer enhancement does not need to exceed the inverse fill fraction. In addition, net gain in overall sensor response is more readily achievable at low target concentrations.

Magnetic Field Effects in Model B12 Enzymatic Reactions. the Photolysis of Methylcob(III)Alamin

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The paradox of multiplex DNA melting on a surface

Under equilibrium conditions, there are two regimes of target capture on a surface--target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to a reverse displacement mechanism of the low affinity species. The melting curve cannot be used in analytical methods to resolve heteroduplexes; only with the simplex system can proper thermodynamics be obtained.

Microarray Temperature Optimization Using Hybridization Kinetics

In any microarray hybridization experiment, there are contributions at each probe spot due to the match and numerous mismatch target species (i.e., cross-hybridizations). One goal of temperature optimization is to minimize the contribution of mismatch species; however, achieving this goal may come at the expense of obtaining equilibrium reaction conditions. We employ two-component thermodynamic and kinetic models to study the trade-offs involved in temperature optimization. These models show that the maximum selectivity is achieved at equilibrium, but that the mismatch species controls the time to equilibrium via the competitive displacement mechanism. Also, selectivity is improved at lower temperatures. However, the time to equilibrium is also extended, so that greater selectivity cannot be achieved in practice. We also employ a two-color real-time microarray reader to experimentally demonstrate these effects by independently monitoring the match and mismatch species during multiplex hybridization. The only universal criterion that can be employed is to optimize temperature based upon attaining equilibrium reaction conditions. This temperature varies from one probe to another, but can be determined empirically using standard microarray experimentation methods.

Real-time optical detection of competitive surface hybridization on microarrays

Application of microarrays for single nucleotide polymorphims (SNPs) has a limited appeal currently due to low reliability of experimental results. Theoretical and experimental studies of surface hybridization of heterozygous samples allow us to identify two factors of observed instabilities. First, reactions may not reach thermodynamic equilibrium in the course of the experiment and second, competitive displacement of low affinity species by high affinity species is the mechanism defining specificity of molecular recognition. Here we describe a real time optical detection arrangement that facilitated the detection of competitive displacement between a wild-type target and a SNP target. Results show that even when the SNP is an order of magnitude lower in concentration (100 pM) then the wild-type target, the kinetics of the SNP hybridization affects hybridization of the wild-type target. Additionally, results show that observed binding kinetics can be altered by adjusting the concentration of the SNP without changing the concentration of the wild-type target. These results have significance when considering what needs to be accounted for when analyzing real time hybridization data.

Theoretical limitations on sensing selectivity in nucleic acid microarrays

Microarray analysis has become increasingly complex due to the growing size of arrays. In this work we explore the effects that temperature and SNP, mismatch, concentration have on the dynamic range of detection in a two component system. A finite element software is used to simulate the mass transport of DNA through a microfluidic chamber to the sensing surface where hybridization of DNA is modeled using the corresponding kinetic equation. We compare the theoretical maximum dynamic range with those from simulations when the match target is 90% of its equilibrium value. Results show that even though the maximum dyamic range decreases as temperature increases the observed dynamic range at 90% match equilibrium grows.

Mass transport effects on real-time nucleic acid microarrays

Microarray analysis has become increasingly complex due to the growing size of arrays. In this work we explore the effects of diffusion and convective fluxes on the time of hybridization and sensing specificity in a single component system. A .nite element software is used to simulate the diffusion of DNA through a microfluidic chamber to the sensing surface where hybridization of DNA is modeled using the corresponding kinetic equation. The differences between diffusion controlled and convection controlled mass transport are investigated as a function of concentration and hybridization time. Hybridization enhancement produced by microfluidics versus stationary diffusion is introduced as a useful metrics for quantitation of mass transport effects.