Hands-on training experience of C-trap

Single molecule manipulation is a powerful tool for studying the behavior of individual molecules, such as DNA, RNA, or proteins. This technique helps us understand the biological mechanisms underlying macroscopic phenomena, particularly in life activities and diseases. Despite its immense utility, single molecule manipulation is difficult and requires extensive training. Mastering these sophisticated experiments can take months or even years. In addition, combining single molecule manipulation with single molecule imaging is highly beneficial but also challenging. The sensitivity of the system makes it difficult to control the signal-to-noise ratio. As a researcher conducting single molecule manipulation experiments, I deeply appreciate its potential and hope for advancements that simplify these experiments and provide additional readouts, such as fluorescent signals.

During the hands-on training program associated with the International Union of Pure and Applied Biophysics (IUPAB) 2024 at the Institute for Quantitative Biosciences at Tokyo University, I was impressed by the C-Trap system developed by LUMICKS. I would like to share my experience with this training and the capabilities of the C-Trap system.

The flight from Singapore to Tokyo takes approximately seven hours, bringing us to the beautiful country of Japan. Our hands-on training began on June 23rd, led by Loïc, an expert on the C-Trap system from LUMICKS. He started with a briefing on the principles of optical tweezers and their significance. One interesting fact I learned in the briefing is that the letter “C” in the name of “C-Trap” is from the last name of one of the founders of LUMICKS who built the very first prototype.

Following this, we moved on to the hands-on training session. The C-Trap system is housed in a separate room and combines optical tweezers, fluorescence microscopy, and microfluidics into one integrated platform. The machine is aesthetically impressive. Loïc began with a demonstration on how to capture a DNA molecule between two beads. The streptavidin-coated beads were first caught in the bead channel, then moved to a channel containing DNA labelled with biotins on both ends. A DNA molecule bound to one of the beads (with one end) is then then flow-stretched, while the other bead is moved to make contact and form a tether.

The method to test if the tether is formed and its quality is impressive. Loïc moved one bead close to the other bead, which had the flowing DNA, and then held it in place for a moment before moving it away. When moving it away, if a resistive force is generated, it indicates that something is connected. But how do we know if it is the desired DNA and if the DNA is in its native state?

They have a pre-calibrated force-extension curve for lambda DNA stretching. If the experimental force-extension curve perfectly aligns with this pre-calibrated curve, it confirms the presence of the correct DNA in its native state. If the curve is shifted, it indicates an issue. For example, if the force increases too quickly with distance, it suggests multiple DNA tethers might have formed. Conversely, if the force doesn’t increase significantly, it means the DNA might be partially broken, with parts of it being single-stranded.

After confirming the correct tether, we switched to the confocal fluorescence channel. Amazingly, we could see the tethered DNA very clearly (Figure 1). I had never done such experiments before, and it was an incredible experience for me. Although I have stretched molecules many times, this was the first time I could literally observe the very molecule I was stretching.

Figure 1  Confocal imaging of the lambda DNA tethered between two beads.

Each of us took turns to perform the same task of finding a proper tether for imaging, and we all succeeded quickly. Loïc then informed us that this was just the beginning, as tethering a single DNA is the most basic application of the C-Trap system. He wanted to challenge us with a more advanced task: creating a smiley face with a tear using the C-Trap system.

This challenge was very interesting, and some of us with experience using optical tweezers in combination with microfluidic devices quickly figured out the method. We positioned the two beads in the flow so that the DNA would be stretched by both the beads and the flow, leading to bending of the DNA. Meanwhile, other DNA molecules were tethered to each of the beads, forming the “tears” (Figure 2).

Figure 2  DNA “smiling with tears”.

To me, this is not only interesting but also highly promising for future experiments. With such sophisticated techniques, many intriguing experiments can be conducted. For example, we could investigate whether the flow affects the movement speed of a protein along the DNA. Additionally, by adjusting the angle of the flow, we could determine if the curvature of the DNA impacts the protein’s movement speed. These questions are both fascinating and significant in the field of mechanobiology.

By the end of the session, Loïc informed us that the C-Trap can manipulate more than two beads simultaneously. He demonstrated that with four laser beams, four beads can be controlled, allowing the formation of two DNA tethers between each pair of beads. Loïc then gave us a final task: to create a “knot” with the two DNA strands using these four beads.

He hinted that the Z position of the beads could also be adjusted, adding another dimension to the challenge.

The idea was straightforward: we needed to lower one pair of beads to allow one DNA strand to go beneath the other, then move it back. After that, we moved one of the beads to let the DNA strands cross and form the knot. Although the operation sounded very complicated, we managed to achieve it in less than 15 minutes, despite it being our first time using the C-Trap (Figure 3).

Figure 3  Make a knot for DNA with C-Trap.

We ended the training session thoroughly amazed by the capabilities of the C-Trap. Although this was my first time working with it, I can envision a lot of interesting work that can be done using the C-Trap. It allows for the manipulation of large biomolecules and the observation of their behavior through fluorescence while applying force to them, addressing important biological questions.

This experience has given me a deep appreciation for the power of the C-Trap. I am immensely grateful for the training opportunity provided by the IUPAB conference. It has broadened my horizons and given me the chance to engage with cutting-edge techniques and meet talented colleagues.