Tension sensor module expression and purification:
- Do not express in HEK 293 cells for more than 48 h as this may lead to accumulation of protein degradation products within cells.
- Purification steps need to be carried out at 4° C to avoid protein degradation.
Preparation of bead-DNA-protein-DNA-bead dumbbell configuration for single-molecule calibration:
- Always work on ice and as quick as possible.
- Buffers should be degassed and filtered to lower the possibility of reactive cysteine crosslinking, oxygen damage, and to reduce contamination with larger particles during measurements.
Testing the oligo-attachment and purification:
- The reactivity of the maleimide-modified oligonucleotides can be tested by reacting the Moligos with dithiothreitol (DTT) followed by a size exclusion chromatography. Having two reactive thiols, makes DTT a perfect mimic for a protein with two reactive cysteins. After letting Moligos react with DTT in the corresponding ratio, all reactive Moligos should form dimers. Due to their doubled size, they can be detected via size exclusion chromatography. Besides estimating the amount of reactive Moligos, the ‘DTT-test’ also allows the optimization of experimental conditions such as reaction time, temperature, TCEP concentration or pH.
- The identification of the correct peak (two Moligos attached to the protein of interest) can be facilitated by a reference run with pure protein.
Optimizing DNA-handle PCR:
- Unreacted anti-sense primers will not be removed by the QiaQuick PCR Purification Kit. This is critical, as these primers carry the sequence complementary to the protein-bound Moligos and, hence, can compete with entire DNA-handles during hybridization. A slightly lower amount of anti-sense primers with respect to both sense primers should help minimizing this risk. Alternatively, unreacted anti-sense primers can be removed by agarose gel electrophoresis and the QiaQuick Gel Extraction Kit.
Evaluating the assembly of protein-DNA chimeras:
- To test the formation of the final protein-DNA chimeras upon mixing of the protein-oligo with DNA-handles, we mix both in various ratios and let them react at RT for about 2 h. Reactions are then tested by agarose gel electrophoresis including a reference, in which only DNA-handles are loaded. The optimal reaction conditions can be assumed for the ladder with the thickest band of double the size of a single DNA-handle. Using dimerized Moligos from the ‘DTT-test’ (see above) allows testing whether the DNA-handles work as they should.
- Each tested pair of beads in each sample chamber needs to be categorized depending on what has been observed, e.g. nothing (= no dumbbell formation), protein with correct DNA-handle length (= successful single dumbbell formation), too short apparent DNA-handle length (= multiple tethers), sticking (= way too many tethers), DNA-handle unzipping (= a force plateau around 15 pN as for DNA hairpins caused by too many DNA-handles used in the sample preparation). The obtained statistics are essential for establishing optimal experimental conditions.
FRET evaluation by fluorescence lifetime microscopy (FLIM):
- Work with physiological expression levels and avoid tension sensor overexpression.
- Adjust laser settings for minimal photo-toxicity and photo-bleaching.
- Only process cells that look healthy; exclude cells that moved during image acquisition.
- Make sure that the temperature remains constant during the experiment (tension sensors are often temperature-sensitive).
- Start the data analysis by performing control experiments and evaluating average FRET efficiencies; evaluate more specialized cases (e.g. spatiotemporal patterns) only after all your control experiments have checked out.
- Always include the donor only control (i.e. YPet fused to the target proteins' N- or C-terminus) and the zero-force FRET control (i.e. tension sensor fused to the target proteins' N- or C-terminus). These controls need to be included in each and every experiment and can not be omitted.
- Check for intermolecular FRET and effects on tension sensor FRET by protein conformation changes.
FRET evaluation by ratiometric imaging:
- Ensure that donor and acceptor images are aligned.
- Potential uneven illumination needs to be corrected.
- Ratiometric FRET measurements are sensitive to experimental settings, like excitation intensity, non-uniform illumination or expression levels of the biosensor.