As it is based on surface-immobilized DNA nanolevers, the switchSENSE® technology is particularly suitable to investigate interactions between proteins and nucleic acids. There are two basic experimental setups that are commonly used to investigate protein/nucleic acids interaction using switchSENSE®:
- Experiments with proteins that target either double-stranded or single-stranded DNA in an unspecific manner (i.e. independent of the DNA sequence), can be easily performed with standard switchSENSE® chips. To achieve sensor electrodes functionalized with double-stranded DNA nanolevers, simply use unmodified complementary DNA as ligand molecule. For experiments with single-stranded DNA, just use X40 buffer as ligand solution.
- To analyze binding to a specific DNA/RNA sequence, a custom sequence can be attached as an overhang to the complementary DNA sequence of the surface tethered ssDNA oligo. The overhang may either be double-stranded or single-stranded. When working with these overhang constructs, it is best to choose a switchSENSE® biochip with 48bp nanolevers. For questions regarding your sequence design, please contact the DBS support team at email@example.com
Protein/nucleic acid interactions generally show an extensive variety of different binding modes that facilitate the interaction.
Due to this complexity, sometimes thorough assay optimization is required to characterize the interaction.
Following, the most common optimization parameters are listed:
- Ionic strength: Many protein/DNA interactions are of ionic nature or ionic forces are required to initiate the interaction. As ionic forces are highly affected by the ionic strength of the buffer solution, the affinity of the interaction might drastically differ for different buffer solutions. At the extreme, this could imply the complete absence of interaction, if the ionic strength is too high, or completely non-specific binding, if the ionic strength is too low.
- pH: Furthermore, ionic forces heavily depend on the charge of the protein of interest. In turn, the protein charge depends on the pH of the experimental buffer solution. Thus, also the pH of the used buffer solution can affect the affinity in a comparable way as the ionic strength.
- Background affinity: Many nucleic acid binding molecules exhibit a certain background affinity to any type of nucleic acid. To differentiate this background affinity from sequence-specific binding, it is crucial to run experiments with a randomized control sequence, preferentially with equivalent base composition. Usually, the background affinity is weaker than the specific interaction, with the consequence that non-specific binding only occurs at comparably high concentrations. In many cases, background affinity can be completely abolished by optimization of ionic strength and/or pH of the buffer solution. If this does not prove successful, the addition of non-specific competitor substances (e.g. fragmented salmon sperm DNA or polydIdC) or other additives (glycerol, heparin) is often highly effective in reducing background affinity.