DNA-driven focusing for protein-DNA binding assays using capillary electrophoresis

A DNA-driven focusing technique is reported for protein-DNA binding assays using capillary electrophoresis. A fluorescent DNA aptamer of 84 nucleotides (RT12) was used to bind to a specific protein, human immunodeficiency virus type 1 reverse transcriptase. The aptamer-protein complexes were effectively focused, separated by capillary electrophoresis, and detected by laser-induced fluorescence (LIF). With this DNA-driven focusing, the separation efficiency of the aptamer-protein complex reached 5 million theoretical plates/m, and the sensitivity for the detection of this complex was improved by 70-120-fold. The DNA-driven focusing technique was further applied to protein-DNA binding assays and to enhance the detection of DNA adducts. DNA adducts present in short oligonucleotides or genomic DNA were recognized by and bound to specific antibodies, and the complexes were focused electrophoretically and detected by LIF. The results demonstrate that the DNA-driven focusing can improve separation, sensitivity, and speed of analysis. The focusing is tolerant to high-salt medium, which is usually necessary to support physiological protein-DNA binding. This technique may be applied to nucleic acid analysis, aptamer affinity analysis, immunoassays for DNA damage, and DNA/RNA based binding assays.