Visualize and measure DNA-protein interactions in real-time at the single-molecule level

Part of: Drug Research

Probe and film DNA-protein interactions in real-time at the single-molecule level

Scientists can use optical tweezers to trap beads and catch a biomolecule, such as DNA, in between. This biomolecule can then be manipulated by moving the beads, while the force and extension are measured. Fluorescently labeled proteins can be visualized with confocal or STED fluorescence microscopy. The combination of optical tweezers with simultaneous multicolor fluorescence measurements allows correlating the mechanical properties of the DNA with the protein activity.

The C-Trap® Optical Tweezers – Fluorescence Microscopy system provides the ability to apply and measure force and extension of the tethered DNA while simultaneously visualizing proteins as they interact with the DNA. In this way, it is possible to film proteins and directly characterize their effect. When DNA-protein interactions are measured at the single-molecule level the exact mechanisms of DNA organization, replication, translation, and repair can be studied in high detail, including conformational changes.

The film in Figure 1 shows the position of bound XRCC4 and XLF on DNA over time at protein concentrations of 5 nM. These are two repair proteins involved in non-homologous end joining which can associate with each other to form complexes capable of bridging DNA. The film shows XLF-XRCC4 protein tandems binding to double-stranded DNA and rapidly diffusing as they are searching for damage sites.

1 Film showing the dynamics of CRCC4-XLF protein complexes on DNA.

Data courtesy of Prof. Erwin Peterman and Prof. Gijs Wuite at the VU University Amsterdam

Quantify and measure the effect of drugs

Scientists can use optical tweezers or acoustic force spectroscopy to trap beads and catch a biomolecule, such as DNA, while an enzyme is interacting with it. It is then possible to study and quantify the effect of small molecules or biologics on the enzyme’s activity. The combination of optical tweezers with simultaneous multicolor fluorescence measurements allows correlation of the mechanical properties of the DNA with the drug activity.

Here, the activity of RNA polymerase (RNAp) was investigated in the presence of different inhibitor (acinetodin, klebsidin, and microscin J25) concentrations. This was done by applying a constant force on the tethered structure to detect the activity of the RNAp in the presence of different known and unknown inhibitor molecules. 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 2 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.

AFS Enzymatic Activity 2
Figure 1 is reprinted with permission from ACS Chem. Bio., 2017, 12 (3), pp 814-824. Copyright 2018 American Chemical Society.

Solutions

C-Trap® Optical Tweezers Fluorescence & Label-free Microscopy
C-Trap

Optical Tweezers and Fluorescence Microscopy

The C-Trap is the world’s first instrument that allows simultaneous manipulation and visualization of molecular interactions in real-time. It combines high-resolution optical tweezers, confocal microscopy or STED nanoscopy with an advanced microfluidics system in a truly integrated and correlated solution.

M-Trap® Optical Tweezers Fluorescence & Label-free Microscopy
m-Trap

Optical Tweezers

The m-Trap is the first entry-level optical tweezers instrument specifically developed for high-resolution single-molecule research. Ultra-high force resolution and stability, with incredible throughput, ease of use and modularity ‒ all at an unprecedented price level.

Key Product FeaturesC-Trap®m-Trap®
DNA-protein-DNA interactions using two DNA strands

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