DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Limits and tradeoffs in the control of autocatalytic systems

Despite the complexity of biological networks, we find that certain common architectures govern network structures. These architectures impose fundamental constraints on system performance and create tradeoffs that the system must balance in the face of uncertainty in the environment. This means that while a system may be optimized for a specific function through evolution, the optimal achievable state must follow these constraints. One such constraining architecture is autocatalysis, as seen in many biological networks including glycolysis and ribosomal protein synthesis. Using a minimal model, we show that ATP autocatalysis in glycolysis imposes stability and performance constraints and that the experimentally well-studied glycolytic oscillations are in fact a consequence of a tradeoff between error minimization and stability. We also show that additional complexity in the network results in increased robustness. Ribosome synthesis is also autocatalytic where ribosomes must be used to make more ribosomal proteins. When ribosomes have higher protein content, the autocatalysis is increased. We show that this autocatalysis destabilizes the system, slows down response, and also constrains the system’s performance. On a larger scale, transcriptional regulation of whole organisms also follows architectural constraints and this can be seen in the differences between bacterial and yeast transcription networks. We show that the degree distributions of bacterial transcription network follow a power law distribution while the yeast network follows an exponential distribution. We then explored the evolutionary models that have previously been proposed and show that neither the preferential linking model nor the duplication-divergence model of network evolution generates the power-law, hierarchical structure found in bacteria. However, in real biological systems, the generation of new nodes occurs through both duplication and horizontal gene transfers, and we show that a biologically reasonable combination of the two mechanisms generates the desired network.

Identification and characterization of the egg-laying hormone from the neurosecretary bag cells of Aplysia

This investigation has resulted in the chemical identification and isolation of the egg-laying hormone from Aplysia californica, Aplysia vaccaria, and Aplysia dactylomela. The hormone, which was originally identified as the Bag Cell-Specific protein (BCS protein) on polyacrylamide gels, is a polypeptide of molecular weight ≈ 6000, which is localized in the neurosecretory bag cells of the parietovisceral ganglion and the surrounding connective tissue sheath which contains the bag cell axons. All three species produce a hormone of similar molecular weight, but varying electrophoretic mobility as determined on polyacrylamide gels. As tested, the hormone is completely cross-reactive among the three species.

Although the bag cells of sexually immature animals contain the active hormone, sexual maturation of the animal results in a 10-fold increase in the BCS protein content of these neurons.

A seasonal variation in the BCS protein content was also observed, with 150 times more hormone contained in the bag cells of Aplysia californica in August than in January. This correlates well with the variation in the animals' ability to lay eggs throughout the year (Strumwasser et al., 1969). There are some indications that the receptivity of the animal to the available hormone also fluctuates during the year, being lower in winter than in swmner. The seasonal rhythm of the other species, Aplysia vaccaria and Aplysia dactylomela, has not been investigated.

A polyacrylamide gel electrophoresis analysis of water-soluble proteins in Aplysia californica revealed several other nerve-specific proteins. One of these is also located in the bag cell somas and stains turquoise with Amido Schwarz. The function of this protein has not been investigated.