Molar Mass Profiling of Synthetic Polymers by Free-Solution Capillary Electrophoresis of DNA−Polymer Conjugates

Abstract

The molar mass distribution of a polymer sample is a critical determinant of its material properties and is generally analyzed by gel permeation chromatography or more recently, by MALDI-TOF mass spectrometry. We describe here a novel method for the determination of the degree of polymerization of polydisperse, uncharged, water-soluble polymers (e.g., poly(ethylene glycol) (PEG)), based upon single-monomer resolution of DNA-polymer conjugates by free-solution capillary electrophoresis. This is accomplished by end-on covalent conjugation of a polydisperse, uncharged polymer sample (PEG) to a monodisperse, fluorescently labeled DNA oligomer, followed by electrophoretic analysis. The monodisperse, charged DNA "engine" confers to each conjugate an equal amount of electromotive force, while the varying contour lengths of the uncharged, polydisperse polymers engender different amounts of hydrodynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a function of the size of the uncharged PEG tail. This provides a profile of the molar mass distribution of the original polymer sample that can be detected by laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE). Theory-based analysis of the resulting electrophoresis data allows precise calculation of the degree of polymerization of the PEG portion of each conjugate molecule. Knowledge of the molecular mass of the uncharged polymer's repeat unit allows for direct calculation of the molar mass averages as well as sample polydispersity index. The results of these analyses are strikingly reminiscent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3.4-, 5-, and 20-kDa nominal average molar mass were analyzed by FSCE and MALDI-TOF; the values of the molar mass averages, Mw and Mn, typically agree to within 5%. Measurements and molar mass calculations are performed without any internal standards or calibration. Moreover, when DNA-polymer conjugate analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min. FSCE offers an alternative to MALDI-TOF for the characterization of uncharged, water-soluble polymers that can be uniquely conjugated to DNA.

Theory of DNA electrophoresis (∼ 1999 –2002 ½)

“…While the electromotive force is the same for all molecules (as each has an identical DNA section), the different lengths of the neutral PEG components give them different mobilities. FSCE compares quite well with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) [96] and reversedphase high performance liquid chromatography (RP-HPLC) [97]; in fact, FSCE was shown to produce separation resolutions approximately five times higher than with the more traditional RP-HPCL, and increase separation efficiencies by 150%. In addition, this method of molar mass profiling has the advantage of not requiring internal standards or calibration, and is amenable to chip-based electrophoretic systems.…”

“…Successful separations of uncharged polymers, based on the same concept, have been achieved, where the DNA was utilized as a uniform, charged "engine" to characterize polydisperse samples of synthetic uncharged polymers [96,97]. Their composite molecules, for example, ssDNA-poly(ethylene glycol) (DNA-PEG) yielded single monomer resolution in free solution CE.…”

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

“…Three different classes of molecules have been considered as potential ELFSE drag-tags, including (i) chemically synthesized polymers [15]; (ii) oligo-N-substituted glycines (oligopeptoids) produced on an automated peptide synthesizer [5,11]; and (iii) natural proteins [12,16]. Chemically synthesized polymers, such as polyethylene glycol (PEG), are not suitable as drag-tags for high-resolution DNA separations, because they are inevitably polydisperse in terms of molecular weight distribution [15]; even a polydispersity index (M w /M n ) of 1.01 is too large for sequencing or genotyping applications.…”

“…Chemically synthesized polymers, such as polyethylene glycol (PEG), are not suitable as drag-tags for high-resolution DNA separations, because they are inevitably polydisperse in terms of molecular weight distribution [15]; even a polydispersity index (M w /M n ) of 1.01 is too large for sequencing or genotyping applications. Oligopeptoids, produced using an automated peptide synthesizer and purified to monodispersity by HPLC, can be useful for the separation of small oligonucleotides, but are not applicable to DNA sequencing with long reads because they are too small.…”

The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

“…This technique cleverly uses an uncharged "label" or "drag" molecule attached to each ssDNA chain in order to break the local balancing between friction and electric force [2-6] which normally leads to comigration of all ssDNA lengths [7,8] (excepting very small fragments [9, 10]) in free solution. More recently, a complementary technique called free-solution conjugate electrophoresis (FSCE) has been used to characterize uncharged, water-soluble polymers that can be uniquely conjugated to ssDNA [11][12][13]. Here, the ssDNA chains are of uniform length, and act as engines to pull the varying lengths of uncharged polymers for electrophoresis leading to single-monomer resolution over a wide range of molecular sizes.…”