Length‐dependent DNA separations using multiple end‐attached peptide nucleic acid amphiphiles in micellar electrokinetic chromatography

Abstract

End-labeled free-solution electrophoresis (ELFSE) is an alternative approach to gel-based methods for size-based electrophoretic separation of DNA. In ELFSE, an electrically neutral "drag-tag" is appended to DNA to add significant hydrodynamic drag, thereby breaking its constant charge-to-friction ratio. Current drag-tag architecture relies on covalent attachment of polymers to each DNA molecule. We have recently proposed the use of micellar drag-tags in conjunction with sequence-specific hybridization of peptide nucleic acid amphiphiles (PNAAs). This work investigates the effect of multiple PNAA attachment on DNA resolution using MEKC. Simultaneous PNAA hybridization allows for the separation of long DNA targets, up to 1012 bases, using micellar drag-tags. Each PNAA handle independently interacts with the micellar phase, reducing the overall mobility of this complex relative to individual PNAA binding. The sequence- and size-based dependence of this separation technique is maintained with multiple PNAA binding over a range of DNA sizes. Results are accurately described by ELFSE theory, yielding alpha=54 for single-micelle tagging and alpha=142 for dual-micelle tagging. This method is the first example of a non-covalent drag-tag used to separate DNA of 1000 bases based on both size and sequence.

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

“…3a) [48,49]. The modified PNAs can bind complementary DNA sequences without any significant perturbation to the hybridization free energy by the presence of the alkyl chain.…”

“…These PNA amphiphiles bind transiently with micelles and vesicles, particularly when bound to a complementary DNA sequence. This allows micelles to be PNA n electrokinetic chromatography, allowing the rapid separation of unlabelled DNA based upon their length and sequence [48,[50][51][52].…”

Modeling and global optimization of DNA separation

“…This problem is of interest to the authors as it was recently discovered by Schneider and co-workers (Grosser et al, 2007; Savard et al, 2008) to be a promising alternative to gel electrophoresis for rapid length based separation of DNA. The optimization model in Eq.…”

“…These methods require that a highly monodisperse drag-tag be attached to the end of the DNAs in the sample. A variation of this technique is micelle-ELFSE electrophoresis (Grosser et al, 2007; Savard et al, 2008), which instead uses ensembles of transiently end-attached surfactant micelles as drag-tags. Here, stochastic variations in micelle size provide a highly uniform drag, despite a polydispersity of micelle size in the overall sample.…”

Microseparation of membrane proteins

“…Since its introduction in the early 1980s, CE has drawn increasing attention of researchers due to its advantages of low sample cost, high separation performance, and ease of system integration and automation. Various CE separation modes have also been developed in the past few decades for enhanced separation selectivity, such as CZE [45,46], MEKC [47,48], capillary sieving electrophoresis (CSE) [49,50], CIEF [51,52], and CEC [53,54].…”