High Resolution Nucleic Acid Electrophoresis by Snagging Mechanisms in Wormlike Micelle Networks

Length-based separations of long kilobase single-stranded DNA (and RNA) is critical to biological research, forensic analysis, and medical diagnostics. DNA separations typically relies on gel electrophoresis, which is labor intensive, suffers from long runtimes of several hours, and has an upper limit of separation due to biased reptation. The Schneider group has developed an alternative method of rapid DNA electrophoresis using surfactant micelle buffers as the separation matrix called micelle tagging electrophoresis (MTE). MTE is capable of extending that biased reptation limit to longer lengths and has been shown to rapidly separate up to 23 kilobases in under 5 minutes. Here, we present a new electrophoresis mechanism in wormlike micelle buffers, using hydrophobic alkane chains and short double-stranded DNA (dsDNA) oligomers to snag on the micelle matrix and hinder mobility for higher resolution separations. Using this strategy, we develop a nucleic acid probe-based assay for viral detection and mRNA vaccine quality control. 

Modification with a hydrophobic alkane chain was done via hybridization of high affinity γ-peptide nucleic acid amphiphiles (γPNAA) probes, which has the added advantage of sequence-specific detection. Using NCBI’s PrimerBLAST and BLAST tools, we identified sequences on 5 viral genomes that would ensure stable and selective binding to its target, judged by their melting temperature T_m. Compared to their predicted and experimental T_m, a lowered binding yield was observed in electrophoresis due to nonequilibrium separation conditions. We determined that a T_m of at least 70°C was necessary to ensure proper probe binding and detection in MTE. The use of these high affinity nucleic acid probes also enable binding in denaturing conditions which is relevant for separations of single-stranded nucleic acids, as well as binding to double-stranded and structured targets by strand invasion. 

We developed an assay for two single-stranded viruses, mouse minute virus (MMV) and murine hepatitis virus (MHV), using γPNAA probes to bind viral genomes and MTE for analysis. We achieved a runtime of under 10 min for separation of both of these genomes, which are 5.1 kilobases and 31.3 kilobases in length for MMV and MHV, respectively. We optimized incubation temperature for maximum probe binding in 5 min to minimize the overall assay readout time. Our initial runs resulted in electropherograms with multiple extraneous peaks, which were removed with the addition of formamide, a denaturant. The elution profile shifts depending on the amount of formamide added and incubation temperature, with more denaturing conditions resulting in delayed elution. Since formamide and high temperature incubation is known to generate structural changes in long single-stranded nucleic acids, we believe these results show that MTE is sensitive to secondary structure. 

We propose a model to describe the migration of ssDNA tagged with a γPNAA probe and a short dsDNA oligomer, which we refer to as a nanosnag. Migration is governed by three independent mechanisms – filtration by the micelle matrix, drag by an attached micelle tag, and snagging of nanosnags in the pore. Hybridization of the γPNAA and the subsequent micelle attachment induce a mechanism where the micelle acts as a drag tag to reduce mobility. Attachment of the nanosnag results in another mobility shift and appears to follow a reptation-like mechanism, where mobility is inversely proportional to nanosnag length. The mobilities of tagged ssDNA can be predicted by a Ferguson plot and a modified Ferguson equation, with additional factors to account for the micelle drag tag and nanosnag reptation. These modifications enable high resolution, rapid analysis of kilobase-length single-stranded nucleic acids that are closely related in length, simply with the addition of a nanosnag for an extra mobility shift. Additionally, these modifications have an added benefit of dramatically improving the peak sharpness and signal strength. 

We present work towards developing a rapid surfactant-based electrophoresis assay for mRNA-LNP quality control. Because the capillary is filled with 1-2 wt% surfactant, mRNA-LNPs can be directly injected into the capillary for mRNA release and separation in a single step. We achieved a separation of a multivalent LNP containing EPO (1109 bases), cre (1609 bases), and βgal (3671 bases) in under 10 minutes. Peaks for mRNA-LNPs also showed a consistent 0.2 min mobility shift compared to naked mRNA, which is thought to be a reflection of the time it takes to release mRNA from the LNP. This mobility shift increases as the surfactant concentration is diluted, possibly because mRNA release is slower with less surfactant in the system. Our group is currently investigating the kinetics of mRNA release by the surfactants used in MTE using a stopped-flow apparatus on a spectrophotometer. Correlation between mobility shifts and kinetic data will provide insight on the applicability of this assay as a method for quality control of LNP formulations. Through the use of simultaneous γPNAA and nanosnag tagging, we can dramatically enhance the resolution of this assay and accurately separate kilobase-length mRNA for quality control and quantitation.