Biomedical subjects

Robert J Meagher

Publications and source records attributed to Robert J Meagher.

9 recordsLinked to original sources

PubMed2006

Free-solution electrophoresis of DNA modified with drag-tags at both ends.

In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.

DNA↗

PubMed2005

New peptidomimetic polymers for antifouling surfaces.

Exposure of therapeutic and diagnostic medical devices to biological fluids is often accompanied by interfacial adsorption of proteins, cells, and microorganisms. Biofouling of surfaces can lead to compromised device performance or increased cost and in some cases may be life-threatening to the patient. Although numerous antifouling polymer coatings have enjoyed short-term success in preventing protein and cell adsorption on surfaces, none have proven ideal for conferring long-term biofouling resistance. Here we describe a new biomimetic antifouling N-substituted glycine polymer (peptoid) containing a C-terminal peptide anchor derived from residues found in mussel adhesive proteins for robust attachment of the polymer onto surfaces. The methoxyethyl side chain of the peptoid portion of the polymer was chosen for its chemical resemblance to the repeat unit of the known antifouling polymer poly(ethylene glycol) (PEG), whereas the composition of the 5-mer anchoring peptide was chosen to directly mimic the DOPA- and Lys-rich sequence of a known mussel adhesive protein. Surfaces modified with this biomimetic peptide-peptoid conjugate exhibited dramatic reduction of serum protein adsorption and resistance to mammalian cell attachment for over 5 months in an in vitro assay. These new synthetic peptide based antifouling polymers may provide long-term control of surface biofouling in the physiologic, marine, and industrial environments.

Biomimetic Materials↗

PubMed2005

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis.

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.

DNA↗

PubMed2005

End-labeled free-solution electrophoresis of DNA.

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.

DNA↗

PubMed2004

A very thin coating for capillary zone electrophoresis of proteins based on a tri(ethylene glycol)-terminated alkyltrichlorosilane.

We describe the use of a tri(ethylene glycol)-terminated alkyltrichlorosilane to create a very thin, protein-resistant "self-assembled monolayer" coating on the inner surface of a fused-silica capillary. The same compound has been demonstrated previously on flat silica substrates to resist adsorption of many proteins. As a covalently bound capillary coating, it displays good resistance to the adsorption of cationic proteins, providing clean separations of a mixture of lysozyme, cytochrome c, ribonuclease A, and myoglobin for more than 200 consecutive runs. Electroosmotic flow (EOF) was measured as a function of pH; the coated capillary retains significant cathodal EOF, with roughly 50% of the EOF of an uncoated capillary at neutral pH, making this coating promising for applications requiring some EOF. The EOF was reasonably stable, with a 2.9% relative standard deviation during a 24 h period consisting of 72 consecutive separations of cationic proteins. Efficiencies for cationic protein separations were moderate, in the range of 190,000-290,000 theoretical plates per meter. The coating procedure was simple, requiring only a standard cleaning procedure followed by a rinse with the silane reagent at room temperature. No buffer additives are required to maintain the stability of the coating, making it flexible for a range of applications, potentially including capillary electrophoresis-mass spectrometry (CE-MS).

Adsorption↗

PubMed2003

Microchannel wall coatings for protein separations by capillary and chip electrophoresis.

The necessity for microchannel wall coatings in capillary and chip-based electrophoretic analysis of biomolecules is well understood. The regulation or elimination of EOF and the prevention of analyte adsorption is essential for the rapid, efficient separation of proteins and DNA within microchannels. Microchannel wall coatings and other wall modifications are especially critical for protein separations, both in fused-silica capillaries, and in glass or polymeric microfluidic devices. In this review, we present a discussion of recent advances in microchannel wall coatings of three major classes--covalently linked polymeric coatings, physically adsorbed polymeric coatings, and small molecule additives. We also briefly review modifications useful for polymeric microfluidic devices. Within each category of wall coatings, we discuss those used to eliminate EOF, to tune EOF, to prevent analyte adsorption, or to perform multiple functions. The knowledgeable application of the most promising recent developments in this area will allow for the separation of complex protein mixtures and for the development of novel microchannel wall modifications.

Acrylic Resins↗

PubMed2002

Multiplexed, high-throughput genotyping by single-base extension and end-labeled free-solution electrophoresis.

Technologies that allow for high-throughput, economical, and accurate single nucleotide polymorphism (SNP) genotyping are becoming crucial for modern genomic efforts. Here, we present a method for multiplexed single-base extension (SBE) genotyping that takes advantage of the unique separation modalities made possible via end-labeled free-solution electrophoresis (ELFSE). Three unique SBE oligonucleotide primers, which probe for mutations of clinical importance in the human p53 gene, were covalently conjugated to three unique polypeptoid frictional end labels and mixed together. This primer-polypeptoid conjugate cocktail was then used in a multiplexed SBE reaction followed by free-solution separation in a 96-capillary array electrophoresis (CAE) instrument. The study was designed to demonstrate multiplexed SNP genotyping of several loci in a single reaction and a single subsequent analysis. Further, the electrophoretic analysis was conducted without any viscous polymeric separation media, was complete in less than 10 min, and can be implemented in any capillary or microfluidic electrophoretic system with four-color fluorescent detection capabilities. Multiplexed SBE-ELFSE genotyping analysis resulted in the simultaneous and accurate genotyping of three p53 loci on five different DNA templates in a single reaction set and single CAE analysis. With the implementation of this method in 96 or more capillaries in parallel, high-throughput screening of SNPs will be accessible to a large number of laboratories.

DNA Primers↗

PubMedDate not supplied

Comblike, monodisperse polypeptoid drag-tags for DNA separations by end-labeled free-solution electrophoresis (ELFSE).

The development of innovative technologies designed to reduce the cost and increase the throughput of DNA separations continues to be important for large-scale sequencing and genotyping efforts. We report research aimed at the further development of a free-solution bioconjugate method of DNA size separation by capillary electrophoresis (CE), in particular, the determination of an optimal molecular architecture for polyamide-based "drag-tags". We synthesized several branched poly(N-methoxyethyl glycine)s (poly(NMEG)s, a class of polypeptoids) as novel friction-generating entities for end-on attachment to DNA molecules. A 30-mer poly(NMEG) "backbone," comprising five evenly spaced reactive epsilon-amino groups, was synthesized on solid phase, cleaved, and purified to monodispersity by RP-HPLC. Three different comblike derivatives of this backbone molecule were created by (1) acetylating the epsilon-amino groups or (2) appending small, monodisperse NMEG oligomers (a tetramer and an octamer). Grafting of the oligo(NMEG)s was done using solution-phase amide bond formation chemistry. Once purified to total monodispersity, the three different drag-tags were studied by free-solution electrophoresis to observe the effect of branching on their hydrodynamic drag or "alpha" and hence their ability to separate DNA. Drag was found to scale linearly with total molecular weight, regardless of branch length. The octamer-branched drag-tag-DNA conjugate was used to separate ssDNA products of 50, 75, 100, and 150 bases in length by free-solution CE in less than 10 min. Hence, the use of branched or comblike drag-tags is both a feasible and an effective way to achieve high frictional drag, allowing the high-resolution separation of relatively large DNA molecules by free-solution CE without the need to synthesize very long polymers.

Acetylation↗

PubMedDate not supplied

Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis.

We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.

Amides↗