Comblike,Monodisperse Polypeptoid Drag-Tags for DNA Separations by End-Labeled Free-Solution Electrophoresis(ELFSE)

摘要

The development of innovative technologies designed to reduce the cost and increase the throughput of DNA separations continues to be important for large-scale sequencing and genotyping efforts.We report research aimed at the further development of a free-solution bioconjugate method of DNA size separation by capillary electrophoresis(CE),in particular,the determination of an optimal molecular architecture for polyamide-based"drag-tags".We synthesized several branched poly(N-methoxyethyl glycine)s(poly(NMEG)s,a class of polypeptoids)as novel friction-generating entities for end-on attachment to DNA molecules.A 30-mer poly(NMEG)"backbone,"comprising five evenly spaced reactive epsilon-amino groups,was synthesized on solid phase,cleaved,and purified to monodispersity by RP-HPLC.Three different comblike derivatives of this backbone molecule were created by(1)acetylating the epsilon-amino groups or(2)appending small,monodisperse NMEG oligomers(a tetramer and an octamer).Grafting of the oligo(NMEG)s was done using solution-phase amide bond formation chemistry.Once purified to total monodispersity,the three different drag-tags were studied by free-solution electrophoresis to observe the effect of branching on their hydrodynamic drag or"a"and hence their ability to separate DNA.Drag was found to scale linearly with total molecular weight,regardless of branch length.The octamer-branched drag-tag-DNA conjugate was used to separate ssDNA products of 50,75,100,and 150 bases in length by free-solution CE in less than 10 min.Hence,the use of branched or comblike drag-tags is both a feasible and an effective way to achieve high frictional drag,allowing the high-resolution separation of relatively large DNA molecules by free-solution CE without the need to synthesize very long polymers.