title: Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.
abstract: Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems.
authors: Department of Chemistry, University of California, Riverside, CA, USA.
authors: Jonathan
authors: J
authors: Ashby
authors: Department of Chemistry, University of California, Riverside, CA, USA.
authors: Samantha
authors: S
authors: Schachermeyer
authors: Department of Chemistry, University of California, Riverside, CA, USA.
authors: Yaokai
authors: Y
authors: Duan
authors: Program in Biomedical Sciences, University of California, Riverside, CA, USA.
authors: Luis A
authors: LA
authors: Jimenez
authors: Department of Chemistry, University of California, Riverside, CA, USA. Electronic address: wenwan.zhong@ucr.edu.
authors: Wenwan
authors: W
authors: Zhong
mesh_terms: D052158:Aptamers, Peptide / Q000737:chemistry; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000737:chemistry*; D031521:Fractionation, Field Flow / Q000379:methods*; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry
chemical_list: D052158:Aptamers, Peptide; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D019809:Streptavidin
keywords: Affinity measurement; Aptamer; DNA folding; DNA–protein interaction; Field-flow fractionation