Monodisperse, “Highly” Positively Charged Protein Polymer Drag-Tags Generated in an Intein-Mediated Purification System Used in Free-Solution Electrophoretic Separations of DNA

Abstract

Free-solution conjugate electrophoresis (FSCE) is a method of DNA sequencing that eliminates the need for viscous polymer solutions by tethering a carefully designed, mobility modifying “drag-tag” to each DNA molecule to achieve size-based separations of DNA. The most successful drag-tags to date are genetically engineered, highly repetitive polypeptides (“protein polymers”) that are designed to be large, water-soluble, and completely monodisperse. Positively charged arginines were deliberately introduced at regular intervals into the amino acid sequence to increase the hydrodynamic drag without increasing drag-tag length. Additionally, a one-step purification method that combines affinity chromatography and on-column tag cleavage was devised to achieve the required drag-tag monodispersity. Sequencing with a read length of approximately 180 bases was successfully achieved with a known sequence in free-solution electrophoresis using one of these positively charged drag-tags. This preliminary result is expected to lead to further progress in FSCE sequencing with ~400 bases read length possible when more “highly” positively charged protein polymers of larger size are generated with the intein system.

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

“…Achieving adequate resolution of all 19 LDR‐FSCE products requires the use of longer drag‐tags with significantly larger hydrodynamic drag as well as a better maleimide linker that would not hydrolyze during temperature cycling. To support this multiplexed separation, a panel of 3 monodisperse “protein polymer” drag‐tags (110, 141, and 204‐amino acids long) was used along with one peptoid (36 mer NMEG) drag‐tag. The drag‐tags were conjugated to the discriminating primers using the heterobifunctional linker sulfo‐SMCC, which has an internal cyclohexane ring that stabilizes the maleimide and prevents ring‐opening hydrolysis.…”

“…These proteins had 18 and 27 repeats of this sequence (141 and 204‐aa total length, respectively). The last method (described thoroughly in ) produced a “highly charged” protein polymer that was 110 amino acids in length. The 110‐aa protein had the amino acid sequence (GTAGSAGTAGSATGAGSAGSRGTAGSGATGASGTGR) 3 ‐GA.…”

“…To achieve resolution of 19 LDR‐FSCE products, longer drag‐tags were necessary than the previously used peptoids. Three practically monodisperse, genetically engineered, highly repetitive “protein polymer” drag‐tags (110, 141, and 204 amino acids in length) were used for this purpose. A more stable linker molecule was used for these drag‐tags to eliminate extra peaks from degradation of the maleimide linker that were seen in the previous LDR‐FSCE separations .…”