Rapid Biosensing Method for Detecting Protein–DNA Interactions

• DOI: 10.1021/acssensors.1c01579
• Publication Date: 2022-01-04T00:28:22Z
• Authors: Shira Roth; Diana Ideses; Tamar Juven-Gershon; Amos Danielli
• Keywords: magnetic modulation biosensing; ∼ 2 h; many biological processes; using magnetic forces; Biosensing Techniques; Biochemistry; short gc; overexpressed buttonhead; play significant roles; significantly advance; based assay ’; magnetic bead; provides unambiguous results; competition experiments; binding occurs; Cancer; experimentally compared; 0303 health sciences; nonspecific binding; current costly; Medicine; attractive alternative method; interacting elements; Biotechnology; limited sensitivity; Chemical Sciences not elsewhere classified; quantitative measures; Biophysics; 612; fluorescent molecule; periodic motion; detectable oscillating signal; magnetic beads; cell lysate; Magnetics; 03 medical and health sciences; rich dna sequences; Virology; Genetics; current techniques; Molecular Biology; Base Sequence; Immunomagnetic Separation; DNA; clinical research; biomedical research; laser beam; cheap components; rapid biosensing method; Physical Sciences not elsewhere classified; based technique; using mmb

Identifying and investigating protein-DNA interactions, which play significant roles in many biological processes, is essential for basic and clinical research. Current techniques for identification of protein-DNA interactions are laborious, time-consuming, and suffer from nonspecific binding and limited sensitivity. To overcome these challenges and assess protein-DNA interactions, we use a magnetic modulation biosensing (MMB) system. In MMB, one of the interacting elements (protein or DNA) is immobilized to magnetic beads, and the other is coupled to a fluorescent molecule. Thus, the link between the magnetic bead and the fluorescent molecule is established only when binding occurs, enabling detection of the protein-DNA interaction. Using magnetic forces, the beads are concentrated and manipulated in a periodic motion in and out of a laser beam, producing a detectable oscillating signal. Using MMB, we detected protein-DNA interactions between short GC-rich DNA sequences and both a purified specificity protein 1 (Sp1) and an overexpressed Buttonhead (BTD) protein in a cell lysate. The specificity of the interactions was assessed using mutated DNA sequences and competition experiments. The assays were experimentally compared with commonly used electrophoretic mobility shift assay, which takes approximately 4-72 h. In comparison, the MMB-based assay's turnaround time is ∼2 h, and it provides unambiguous results and quantitative measures of performance. The MMB system uses simple and cheap components, making it an attractive alternative method over current costly and time-consuming techniques for analyzing protein-DNA interactions. Therefore, we anticipate that the MMB-based technique will significantly advance the detection of protein-DNA interactions in biomedical research.