Design and Production of Protein-Based Polymers for Application as Drag-Tags in Free-Solution DNA SequencingPublic Deposited
End-Labeled Free-Solution Electrophoresis, or ELFSE, is an alternative strategy for DNA sequencing that proposes to eliminate the need for a viscous sieving matrix for size-based DNA separation. In this bioconjugate method, a perturbing entity or "drag-tag" is attached to differently sized DNA fragments produced by the Sanger reaction. This drag-tag alters the overall charge-to-friction ratio so that DNA separation by size can be accomplished by free-solution electrophoresis. Rapid separations with long read lengths are theoretically possible using ELFSE. Application of ELFSE to integrated "lab-on-a chip" microfluidic devices currently under development is facilitated by the absence of a viscous polymer solution. For successful DNA sequencing, this perturbing entity needs to be large, water-soluble, preferably uncharged, monodisperse, and also have a point for unique attachment to DNA. A non-natural repetitive polypeptide, or "protein polymer", has the potential to meet these numerous requirements for optimal ELFSE performance. A small (127 amino acids) drag-tag was previously used to demonstrate that ELFSE sequencing is possible using a protein polymer as the drag-tag. Obtaining a sufficiently large and monodisperse protein polymer drag-tag has been the focus of this research and achieving this goal proved to be more challenging than originally expected. Charged and uncharged protein polymers of varying lengths were evaluated for their potential as drag-tags. Sequences incorporating additional hydrophobic residues were also investigated as well as an alternative purification strategy using self-cleaving affinity tags. Proteins expressed with an N-terminal affinity tag were compared to those expressed with a C-terminal affinity tag to determine the best method for obtaining monodisperse protein polymers. The biophysical properties of these drag-tags were measured via spectroscopy. Extensive investigation into aspects of protein polymer design, production, and purification was required to finally produce a sufficiently monodisperse drag-tag of double the previous size, bringing us closer towards the goal of achieving long-read ELFSE sequencing.