Electrophoresis of megaDalton proteins inside colloidal silica.

With the growth of the biopharmaceutical industry, there is a need for rapid size-analysis of proteins on the megaDalton scale. The large pore sizes needed for such separations cannot be easily reached by pushing the current limits of size-exclusion chromatography or gel electrophoresis. The concept detailed here is the formation of arbitrarily wide pores by packing nonporous colloidal silica in capillaries. This method can be called packed-capillary electrophoresis, or "pCE". Electrophoresis of protein standards (11-155 kDa) by pCE, using 345 nm diameter particles in 100 µm diameter capillaries, gives 2x higher resolution than a typical PAGE gel in 1/6 of the time. The electropherograms show that pCE is highly efficient, with half-micrometer plate heights for all seven standards, giving 105 plates for a 50 mm length. The large pore radius of 65 nm enables baseline resolution of proteins of 0.72, 1.048 and 1.236 MDa in less than 15 min. The short separation time of pCE is attributed to the absence of small pores that restrict protein migration in gels. The pCE separation is applied to the analysis of a stressed pharmaceutical-grade IgG4 sample, giving unprecedented baseline resolution of monomer, dimer, trimer and tetramer in less than 10 min.

A near-infrared, surface-enhanced, fluorophore-linked immunosorbent assay.

The enzyme-linked immunosorbent assay is commonly used for research and clinical applications but typically suffers from a limited linear range and is difficult to multiplex. The fluorophore-linked immunosorbent assay is a closely related technique with good linear range and the ability to detect multiple antigens simultaneously but is typically less sensitive. Here, we demonstrate a near-infrared, surface-enhanced fluorophore-linked immunosorbent assay with sensitivity comparable to its enzyme-linked counterpart. A 59-fold enhancement to sensitivity (slope of linear fit) and an 8-fold improvement in LOD are demonstrated on a direct assay with rabbit immunoglobulin-G as a model system. The technique is also tested on a clinically relevant assay to detect alpha-fetoprotein, in which a 42-fold enhancement to sensitivity is demonstrated along with a 16-fold improvement in LOD. The technique enables these accomplishments while maintaining the entire traditional assay protocol and simply adding two steps at the end. This technique may prove superior to current protocols for biomarker research and clinical diagnoses, which require high sensitivity along with quantitation over an extended range.

Capillary electrophoresis Western blot using inkjet transfer to membrane.

Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 - 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, ß-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.

Fluorescent structural DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates.

Highly labeled DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates (dNTPs) were developed as a source of dNTPs for DNA polymerase. The particles were prepared by strand-displacement polymerization from a self-complementary circular template. Imaged by atomic force microscopy, these functionalized particles appear as condensed fuzzy balls with diameters between 50 and 150 nm. They emit a bright fluorescent signal, detected in 2 ms exposures with a signal-to-noise ratio of 25 when imaged using a TIR fluorescence microscope.

An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces.

Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis.

Fast Immunoassay for Microfluidic Western Blotting by Direct Deposition of Reagents onto Capture Membrane.

Western blotting is a widely used protein assay platform, but the technique requires long analysis times and multiple manual steps. Microfluidic systems are currently being explored for increased automation and reduction of analysis times, sample volumes, and reagent consumption for western blots. Previous work has demonstrated that proteins separated by microchip electrophoresis can be captured on membranes by dragging the microchip outlet across the membrane. This process reduces the separation and transfer time of a western blot to a few minutes. To further improve the speed and miniaturization of a complete western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed. Flow deposition of antibodies is used to overcome diffusion limited binding kinetics so that the entire immunoassay can be completed in 1 h with detection sensitivity comparable to incubation steps requiring 20 h. The use of low microliter/min flow rates with antibody reagents applied directly and locally to the membrane where the target proteins have been captured, reduced antibody consumption ~30-fold. The complete western blot was applied to the detection of GAPDH and ß-Tubulin from A431 cell lysate.

Correlation between scattering properties of silver particle arrays and fluorescence enhancement.

We report on the nanofabrication of patterned silver particle arrays using electron-beam lithography and the evaluation of their optical properties using backscattering and fluorescence spectroscopy. The silver particles varied in size from 100 to 250 nm and were in the shape of circles, squares, and triangles. Three inter-particle separations, 40, 65, and 90 nm as measured from the side of one particle to the side of the next particle, were used. We observed distinctive patterns of backscattering and fluorescence intensity depending on the particle size, inter-particle spacing, and excitation/emission wavelength used. Our approach allows for a study of the correlation between the backscattering intensities and fluorescence enhancement of silver particle arrays, which can be used to optimize the arrays for multi-fluorophore configuration for advanced sensing designs.

Synthesis and stability of novel terminal phosphate-labeled nucleotides.

Novel compounds consisting of a nucleotide triphosphate labeled with a PEG linker and various terminal groups attached to the gamma-phosphate of the nucleotide were constructed for use in efforts to produce a new class of DNA sequencer. The stability of these novel compounds was investigated to determine their utility as sequencing reagents. Hydrolysis rate constants were measured for both the natural nucleoside triphosphate dATP and novel dATP derivatives. The gamma-labeled dATP was approximately 20-fold more stable to hydrolysis than dATP.

Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity.

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.

Incorporation of reporter-labeled nucleotides by DNA polymerases.

The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA.

In vitro production and screening of DNA polymerase eta mutants for catalytic diversity.

Mutant DNA polymerases have become an increasingly important tool in biotechnology. The ability to examine the activity and specific properties of enzymes has a crucial role in the characterization of the enzyme. We have developed several systems for characterizing DNA polymerases that combine random mutagenesis with in vivo selection systems. However in vivo screening systems for specific properties are sometimes unavailable. The ability to quickly screen for polymerase activity has many applications, including the identification of compounds that can inhibit polymerase activity, identifying the properties of newly discovered polymerases, and engineering new biological properties into existing polymerases. These applications can both expand the knowledge of the basic science of polymerases and can further industrial efforts to identify new drugs that specifically target polymerase activity. Here we present a high-throughput in vitro assay to select for active polymerases. We show the applicability of this assay by measuring the level of activity for a set of in vitro synthesized polymerase mutants and by screening for the incorporation of a fluorescent nucleotide analog by DNA polymerases.

Viral error catastrophe by mutagenic nucleosides.

Riboviruses and retroviruses have the highest rates of mutations of any known organism. Increasing the mutation rate of these viruses could exceed the error threshold for viability of a viral population within a host. Recent experiments with mutagenic nucleoside analogs validate this new approach to treating infection of RNA viruses. Lethal mutagenesis with HIV-infected cells in culture has been documented and has been postulated to be the mechanism for treatment of hepatitis C with ribavirin. We consider the viral dynamics involved in the formation of a quasispecies, the choice of mutagenic nucleoside analogs, and the studies that have demonstrated the feasibility of lethal mutagenesis.

Multiple mutations and cancer.

Most human tumors are highly heterogenous. We have hypothesized that this heterogeneity results from a mutator phenotype. Our premise is that normal mutation rates are insufficient to account for the multiple mutations found in human cancers, and, instead, that cancers must exhibit a mutator phenotype early during their evolution. Here, we examine the current status and implications of the mutator phenotype hypothesis for the prognosis, treatment, and prevention of human cancers.

Predicting demographic group structures based on DNA sequence data.

The ability to infer relationships between groups of sequences, either by searching for their evolutionary history or by comparing their sequence similarity, can be a crucial step in hypothesis testing. Interpreting relationships of human immunodeficiency virus type 1 (HIV-1) sequences can be challenging because of their rapidly evolving genomes, but it may also lead to a better understanding of the underlying biology. Several studies have focused on the evolution of HIV-1, but there is little information to link sequence similarities and evolutionary histories of HIV-1 to the epidemiological information of the infected individual. Our goal was to correlate patterns of HIV-1 genetic diversity with epidemiological information, including risk and demographic factors. These correlations were then used to predict epidemiological information through analyzing short stretches of HIV-1 sequence. Using standard phylogenetic and phenetic techniques on 100 HIV-1 subtype B sequences, we were able to show some correlation between the viral sequences and the geographic area of infection and the risk of men who engage in sex with men. To help identify more subtle relationships between the viral sequences, the method of multidimensional scaling (MDS) was performed. That method identified statistically significant correlations between the viral sequences and the risk factors of men who engage in sex with men and individuals who engage in sex with injection drug users or use injection drugs themselves. Using tree construction, MDS, and newly developed likelihood assignment methods on the original 100 samples we sequenced, and also on a set of blinded samples, we were able to predict demographic/risk group membership at a rate statistically better than by chance alone. Such methods may make it possible to identify viral variants belonging to specific demographic groups by examining only a small portion of the HIV-1 genome. Such predictions of demographic epidemiology based on sequence information may become valuable in assigning different treatment regimens to infected individuals.