Nanopore sensing of individual transcription factors bound to DNA

Abstract

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes.

Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

“…The original plasmid for Egr---1 (DNA binding domain) was kindly provided by Dr. Scot Wolfe. The protein was expressed and purified as previously described (50), by cloning it into a pGex2t plasmid (GE Healthcare Life Sciences) along with a cleavable glutathione S---transferase (GST) tag. Fusion protein was expressed in BL21 cells grown in LB media at 37 °C, and purified using a glutathione sepharose column (GE Healthcare Life Sciences).…”

Relevance of the Drag Force during Controlled Translocation of a DNA–Protein Complex through a Glass Nanocapillary

“…63 The crossover point at 300 mM KCl is attributed to the dominant frictional forces between the ions and DNA, causing a current drop. 63 In our case, both DNA and protein 15,20,21,64 In our system we detected current signatures of DNA−protein complexes in physiological conditions with the concentration of KCl in the range of 40− 150 mM (Figure 4b,c,d). Notably, the current in addition to the force signal increases the resolution for detection of local protein structures on DNA, especially in nanocapillaries of small diameters ( Figure S8).…”

Nano‐Optical Tweezing of Single Proteins in Plasmonic Nanopores

“…More specifically, biological and solid‐state nanopores are nanoscale openings in a thin membrane that electrophoretically transport biomolecules toward the sensing region via an externally applied bias voltage. This active transport results in great sensor response times and has been used in the past decade to characterize protein, DNA, and protein–DNA interactions . However, the mode of transport unavoidably leads to an uncontrollably fast passage of the molecules through the sensing region and high translocation speeds that lead to a limited temporal resolution in the ionic‐current readout .…”

“…This active transport results in great sensor response times and has been used in the past decade to characterize protein, [11,12] DNA, [13] and protein-DNA interactions. [14,15] However, the mode of transport unavoidably leads to an uncontrollably fast passage of the molecules through the sensing region and high translocation speeds that lead to a limited temporal resolution in the ionic-current readout. [9] Some strategies to reduce this fast speed have been employed, [16,17] but these require labeling and failed to demonstrate full external motion control.Alternatively, plasmonic nanotweezers have demonstrated the capacity to retain small particles and biomolecules in their sensing volume indefinitely [18][19][20][21] through optical trapping.…”