Multimodal electrochemical sensing of transcription factor-operator complexes

• Published in: Analyst (London) Vol. 139; no. 6; pp. 1463 - 1471
• Main Authors: Williams, Keeshan, Kim, Chung-Sei, Kim, Jin Ryoun, Levicky, Rastislav
• Format: Journal Article
• Language: English
• Published: England 21.03.2014
• Subjects: Bacteriophage lambda - chemistry; Bacteriophage lambda - metabolism; Base Sequence; DNA, Viral - chemistry; DNA, Viral - metabolism; Electrochemical Techniques - instrumentation; Equipment Design; Molecular Sequence Data; Oxidation-Reduction; Repressor Proteins - metabolism; Viral Regulatory and Accessory Proteins - metabolism
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c3an02128a

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. Changes in diffusive movements, surface potential, and interfacial impedance of DNA monolayers are combined to analyze binding of unlabeled transcription factors.