Shane T. Grosser — 2005-06 Fellow
The simplest and perhaps most common implementation of capillary electrophoresis (CE), capillary zone electrophoresis, is relatively inefficient at separating DNA targets. This is primarily due to the nearly constant charge-to-mass ratio, or more specifically, the charge-to-friction ratio that DNA possesses. As a result of this symmetry, the electrophoretic mobility, or the velocity that a DNA target migrates through a given electric field, is largely insensitive to both its length and sequence. To circumvent this issue, a number of operational modes of CE can be utilized, but the majority of these techniques rely on breaking the charge-to-friction symmetry of the DNA target.
We have chosen to break this ratio through the interaction of a DNA target with a self-assembled micellar structure, thereby increasing the hydrodynamic drag experienced by the DNA target, without appreciably changing its charge. Although DNA shows no inherent interaction with a non-ionic micelle, we have chosen to exploit the amphiphilic nature of a peptide nucleic acid amphiphile (PNAA) probe, conferred through the addition of an 18 carbon aliphatic chain, to promote DNA/micelle interaction. The unique interaction of the PNAA/DNA duplex with a micellar subphase can be leveraged in micellar electrokinetic chromatography (MEKC). MEKC is an operational mode of CE in which a surfactant is added to the running buffer at a concentration suitable for micelle formation. Separation of an analyte becomes dependant on both the intrinsic electrophoretic mobility of the analyte, and its interaction with the micellar pseudostationary phase. By employing a non-ionic surfactant as the pseudostationary phase, we have demonstrated the ability to sequence specifically separate single stranded DNA targets up to 450 bases in length. Although we are confident in our ability to separate significantly longer DNA targets, we are currently unable to generate sufficiently long single stranded DNA targets.
In an effort to circumvent this issue, we have recently investigated the synthesis of DNA based amphiphiles. The covalent attachment of a hydrophobic moiety to a DNA oligomer would allow the implementation of enzymatic replication (PCR) and subsequent electrophoretic separation of both single and double stranded DNA. The amplification of long DNA targets is straightforward using PCR and the subsequent analysis in MEKC is likely to be analogous to the PNAA system previously studied. Covalent attachment of aliphatic tails has been accomplished by DMAP activation of a 5’ terminal phosphate and subsequent reaction with octadecylamine. Experiments show that implementation of a DNA amphiphile in MEKC is straightforward and could have potential implications in the fields of DNA sequencing and single nucleotide polymorphism analysis.