Study and Visualize DNA Transcription Mechanisms at the Nanoscale

Part of: DNA Transcription

Visualization of DNA-protein interactions

In this experiment, a DNA molecule is tethered between two beads while multiple fluorescently labeled proteins are interacting with it. We can visualize these interactions and track them over time using a multicolor confocal or STED fluorescence microscopy. The resulting kymograph unveils the number, position, diffusion and (un)binding events of the proteins along the DNA.

C-Trap DNA-Protein Interactions General

Figure 1 shows the fast dynamics of TFAM proteins on DNA at high protein density. The kymograph was recorded using two methods: confocal (left) and STED (right). The use of STED enabled the tracking of individual protein trajectories, including (un)binding and oligomerization events, which were not always observable with the diffraction-limited resolution of the confocal microscopy.

C-Trap STED Comparison

1 Kymograph of the dynamics of human TFAM-Atto 647N at ~5 nM on λ-DNA at a constant force of 4 pN using confocal microscopy (left) and STED microscopy (right).

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

Heller et al. (2013)
Nature Methods

Investigation of the activity and states of DNA replication motor proteins

A DNA molecule is caught and tethered between a bead and a single DNA polymerase (DNAp) replicating it. We can calculate the DNAp activity as it replicates the DNA, by stretching the DNA strand with a constant force and measuring the distance between the beads.

C-Trap DNA Transcription

Figure 2 shows measured data of the activity of T7 DNA polymerase. This protein participates in DNA replication and has 3’ to 5’ exonuclease activity, which is enhanced in the presence of force. Optical tweezers hold the DNA construct (8.3 kbp), tethered between two beads (ø=1.86 mm) at a constant force of 45 pN to observe force-induced exonucleolysis at the single-molecule level. Short activity bursts ranging between 3 and 10 nucleotides are revealed, interspersed by frequent pauses of varying duration.=

Single-molecule measurements of stepping behavior of biomolecular motors along nucleic acids will supply important new information about their enzymatic mechanisms. Next to this experiment, the multicolor fluorescence detection allows quantifying the FRET efficiency changes in time and enables correlating a specific conformational change with activity bursts of DNAp.

C-Trap T7 Polymerase

2 Activity bursts of DNA polymerase performing force-induced exonucleolysis on an ssDNA.

Measurement of riboswitch conformational changes

Here, an RNA polymerase (RNAp) is caught and tethered between a bead and the nascent RNA strand. As the nascent RNA emerges from the RNAp the molecule begins to fold immediately forming riboswitches. We can follow the states and activity of the formed riboswitch, by stretching the tether with a constant force and measuring the distance between the beads.

Co-translational Step Optical Tweezers Fluorescence Microscopy

Figure 3 shows the results of an experiment where we stretched and relaxed the RNA molecules with a constant load (10 nm/s, blue), while recording the force-extension curves. At approximately 8 pN we observe an unfolding rip, approximately 15 nm long. When we relaxed the molecule the refolding curve followed the same path as the unfolding, which corresponded to a rip of similar size. This shows that the unfolding of the RNA molecule is a reversible process. Cycles of unfolding and refolding could be repeated multiple times with the same result.

In the second experiment, we evaluated the structural transitions of our RNA target molecule over time. To this end, we brought a single RNA molecule to a specific pretension and studied its transitions. Figure 4 shows two major states at tensions around 6.2 pN and 7.2 pN, while a third conformational state was also occasionally accessed.

Force-distance curves corresponding to the extension (red, 10 nm/s) and retraction (blue, 10 nm/s) of a single RNA tether.

3 Force-distance curves corresponding to the extension (red, 10 nm/s) and retraction (blue, 10 nm/s) of a single RNA tether.

C-Trap RNA Unfolding

4 two-second time trace of a single RNA molecule held at an initial tension of ~7 pN. Dashed black lines show different possible visited states.

Sample courtesy of Prof. Hang Shi at Tsinghua University.

Solutions

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 and Fluorescence Microscopy

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™
co-Localization studies
Visualization of binding position and diffusion of proteins
Enzymatic activity that alters DNA/RNA mechanics (e.g. exonucleases, DNAp, helicases, SSB)
Single-molecule FRET studies
Rapid buffer exchange for fast experimental workflow
Conformational changes and states

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