Abstract
DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show - through the use of biotinylated DNA and streptavidin - that this selective adsorption extends to DNA-protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.
Highlights
Interactions between DNA and proteins regulate a number of processes crucial to cellular function that include transcription, chromosome maintenance, DNA replication and repair
They can result in non-specific protein–surface interactions, which are non-trivial to deconvolute from specific DNA–protein interactions
For atomic force microscopy (AFM) studies of streptavidin binding, a 672 bp length of DNA was prepared by PCR amplification of a section of lambda DNA (New England Biolabs) using a forward primer 5′-CGATGTGGTCTCACAGTTTGAGTTCTGGTTCTCG-3′ and reverse primer 5′-GGAAGAGGTCTCTTAGCGGTCAGCTTTCCGTC-3′ purchased from Integrated DNA Technologies
Summary
Interactions between DNA and proteins regulate a number of processes crucial to cellular function that include transcription, chromosome maintenance, DNA replication and repair. Graft-copolymers with a cationic PLL backbone and PEG side chains (PLLg-PEG) have proven effective at self-assembling into densely packed polymeric brushes to form non-fouling surfaces.[23,24,25,26,27,28] In addition, bio-recognition sites, such as RGDpeptides, have been incorporated into these films to promote cell adhesion whilst suppressing the non-specific adsorption of serum proteins.[29,30] the incorporation of biotinterminated PEG chains has been used to form small molecule biosensors that selectively bind streptavidin, neutravidin and avidin.[31] Unmodified PLL-g-PEG films have shown the ability to selectively adsorb DNA polyelectrolytes onto the underlying positively charged PLL layer, whilst the PEG layer remains impervious to other proteins, as confirmed by fluorescence imaging.[28]
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