title: Multimodal electrochemical sensing of transcription factor-operator complexes.
abstract: Interactions of proteins with nucleic acids arise at all levels of cellular function, from chromosomal packing to biological regulation. These interactions can be analyzed in a high-throughput fashion by immobilizing the DNA sequences of interest, possibly numbering in the thousands, at discrete locations on a solid support and identifying those sequences that a protein analyte binds. Ideally, such surface assays would use unlabeled analyte to simplify protocols and avoid the possibility of perturbing the protein/DNA interaction. The present study compares three electrochemical modalities for simultaneously detecting binding of unlabeled transcription factor proteins to immobilized DNA duplexes based on (i) changes in the duplex diffusive motions, (ii) variations in the surface potential, and (iii) variations in the interfacial charging impedance, all of which can be conveniently derived from AC voltammetry traces. Cro protein from bacteriophage lambda is used as a model transcription factor. Specific binding of protein was successfully detected through modalities (i) and (ii), but not (iii). The effectiveness of these techniques is compared as a function of sampling frequency and protein concentration. Binding of 15 kDa Cro slowed down rotational diffusion of immobilized duplexes approximately 3-fold, and induced up to 5 mV changes in the surface potential. Moreover, by assessing Cro binding to bacteriophage operators of variable affinity, the study illustrates how contrast between specific and nonspecific interactions impacts detection.
authors: Department of Chemical & Biomolecular Engineering, Polytechnic Institute of New York University, Brooklyn, New York 11201, USA. rl1306@nyu.edu.
authors: Keeshan
authors: K
authors: Williams
authors: Chung-Sei
authors: CS
authors: Kim
authors: Jin Ryoun
authors: JR
authors: Kim
authors: Rastislav
authors: R
authors: Levicky
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000295:instrumentation*; D004867:Equipment Design; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012097:Repressor Proteins / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins