Multiplexed force extension and manipulation of DNA-protein interactions
Here, multiple protein-coated DNA molecules are tethered between a bead and a glass surface. Using the AFS we can stretch the DNA molecules by pulling the beads away from the surface while measuring the z-position of each individual bead. This makes it possible to obtain the force-distance curve of many protein-coated DNA molecules in parallel.
Figure 1 shows the force-extension curve of a DNA molecule measured before (left) and after (right) the incubation of 1 μM of RecA – a protein involved in DNA repair. From the figure we can observe that RecA substantially lengthens the DNA as it forms filaments around the DNA structure, preventing it from coiling.
Figure 2 shows the normalized length-time traces of two individual DNA molecules in the presence of 0.5 μM RecA. At a constant force of 40 pN, the DNA length increases to >1.4x the contour length (Lc) because of RecA binding to the DNA. When the force is set to 2.5 pN again, the length of the DNA decreases due to RecA disassembly. This indicates that the RecA binding is strongly dependent on tension and is therefore enhanced by increased force. From the figure, we can also observe a slightly different behavior between the two molecules which underlies the importance of obtaining many single-molecule measurements.
Highly parallel measurements of DNA-protein interactions typically require that both constant and dynamic forces can be applied on the DNA. A high force and distance resolution and the ability to apply hundreds of picoNewtons to the DNA molecule are necessary to obtain the complete force-distance curve.
Investigation of protein activity involved in DNA Replication
Multiple DNA tethers are attached at one end to the surface of the AFS chip and at the other to a polystyrene bead through RNA polymerase (RNAp) stalled on the DNA template. As the position of the bead changes proportional to the position of the protein on the DNA, we can study the enzymatic activity of the proteins by measuring the z-position of the beads while keeping the force constant.
Figure 3 shows measurement data from a proof-of-concept experiment involving DNA molecules with stalled E. coliRNA polymerase (RNAp). Once transcription and replication are initiated we can precisely monitor the activity of many individual E. coli RNAp molecules in parallel by measuring the DNA elongation in real time.
The graph shows the typical complex nature of the protein activity: stochastically occurring elongation is frequently interrupted by pausing events of different nature.
Investigation of the effect of inhibitors on enzymatic activity
Here, a single DNA tether is attached at one end to the surface of the AFS chip and at the other to a polystyrene bead through RNAp stalled on the DNA template. Next, constant force is applied to detect the activity of the RNAp in the presence of different known and unknown inhibitor molecules.
In practice, the effect of two novel peptides, acinetodin and klebsidin, was investigated with respect to transcription elongation generated by RNA polymerase. The detection of transcription elongation was determined by the presence of varying concentrations of acinetodin, klebsidin and microcin J25; the last peptide being a known transcription inhibitor. Figure 4 shows that – just as microsin J25 – both acinetodin and klebsidin inhibit transcript elongation by E. coli RNAp, with the inhibitor activity of klebsidin being comparable to that of microcin J25 and more active than the activity of acinetodin.
The same experiment can be done with many single-molecules in parallel allowing you to collect properties, such as the rate and pausing events of each RNAp enzyme in singulo, and map their collective distribution in histograms. In this manner it is possible to identify the processivity properties of RNAp in the presence of different molecules leading to new insights.
4 Representative elongation profiles for individual RNAp’s for various concentrations of microcin J25, klebsidin, and acinetodin and in the absence of inhibitors plotted as nucleotides transcribed vs time. Data were filtered by a 0.5 Hz low pass filter.