DRX
Specific Experimental Considerations
As the DNA switching process is extremely sensitive to changes in the hydrodynamic drag, minor changes in the molecular structure of proteins and other macromolecules can be detected as changes in the switching dynamics. A typical application of such an experiment, is the detection of ligand-induced conformational changes, for example upon association of a small molecule to a protein.For the successful detection of a conformational change, please consider the following aspects:
- The molecule of interest can be captured via hybridization after covalent coupling, or via a tag system
(His6-Tag, Strep-Tag, biotin-streptavidin interaction, etc.) - Significant conformational changes can be resolved as changes of a protein’s hydrodynamic diameter, which is calculated using the Lollipop model. Please note that this kind of analysis is only available when using appropriate buffer conditions.
Also see “What is the Lollipop Model?” - Please analyze changes in the hydrodynamic diameter using the “Sizing” evaluation tool in switchANALYSIS.
- Often, conformational changes affect only minor portions of the protein molecule, which might not be resolvable by use of the Lollipop model.
- Nevertheless, these changes usually still change the hydrodynamic properties of the protein, resulting in changes in the switching speed.
- These changes can be evaluated as relative Dynamic Response change in comparison to the bare DNA reference measurement.
- For such analyses, please use the “Conformational Change” evaluation tool in switchANALYSIS.
- It is advisable to use a detergent-free running buffer (e.g. PE40 without Tween-20).
- Liposomes can be immobilized on the biosurface using a cholesterol-ligand, i.e. cholesterol modified DNA nanolever
(cholesterol intercalates into lipid bilayer). - Use low flow rates for association and dissociation (e.g. < 50 µl/min) to prevent destabilization of membranes.
- Commonly used liposome concentration for sufficient surface coverage: 300 µl of a 0.2 mM solution (phospholipid concentration)
In some cases, experiments with peptides can be challenging due to the chemical properties of the individual peptides. Major aspects that can negatively affect your experiments and suggested solutions for troubleshooting are listed below:
Peptide solubility:
A frequent problem in such experiments is the low solubility in water of some peptides. This can be improved by addition of a compatible organic solvent to both the running and the sample buffer used in your switchSENSE® experiment. A commonly used solvent is dimethyl sulfoxide (DMSO) which may be supplemented to the experimental buffer up to 5 % v/v.
Peptide charge:
If a peptide contains charged amino acids, it is likely to exhibit a large mass to charge ratio which can prove to be problematic, especially when working with charged surfaces. The consequences range from extensive unspecific surface adsorption to the absence of specific binding due to electrostatic repulsion. If you observe such a behavior, try to increase to salt concentration of your running and sample buffer. This will increase the shielding of the charges of the peptide molecule.
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 support@dynamic-biosensors.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.
switchSENSE® measurements can be performed from 8 to 70°C at constant temperatures.
Additionally, the temperature of the biochip can be ramped up or down (1-10°C/min), e.g. during a melting experiment.
Be aware that the chip life time may suffer from long experiments at elevated temperatures and by repeated heat/cool cycles.
Generally, switchSENSE® can tolerate measurements in a variety of complex matrices, including cell lysates and high serum concentrations up to 80%. Notably, the robust fluidic system, which DRX instruments are equipped with, allows to handle rather complex solutions.
Nevertheless, measurements in complex media usually require a certain degree of assay development. In most cases, this is owed to the occurrence of unspecific adsorption to surfaces of the sensor system.
For successful experiments, please consider the following aspects:
- Always filter samples or remove solid components by centrifugation prior to injection.
- The addition of non-specific competitor substances (fragmented salmon sperm DNA, polydIdC) is usually required to reduce background affinity.
- When using salmon sperm DNA or similar reagents, make sure to use a well buffered solution to avoid acidification.
- Measurements in static mode are usually more robust when complex media are tested.
- The activity of nucleases that are potentially present in the test solution, is often reduced by EDTA.
- Always perform a reference injection on DNA nanolevers without ligand.
- As complex media usually differ significantly in composition, usually the ideal dilution of the original sample must be optimized.