Visual biochemistry of protein-nucleic acid interactions using a multi-user single-molecule optical trapping fluorescence microscope.

To study complex biological systems, biochemists often take a reductionist experimental approach: biomolecules are individually purified and recombined "in a test tube" and their interactions measured. Although these ensemble experiments are the cornerstone of biochemical study and can reveal much about biomolecular function, they are often hard to interpret. The mathematical rules used to quantify the interactions rely on synchronisation between molecules assumed to have identical properties. However, because the solutions contain billions of molecules, the processes being measured can become desynchronised. A protein population can have differences in activity (static disorder) or individual proteins may alter their activity with time (dynamic disorder). Additionally, some physical properties are hard to manipulate, such as forces acting on a biomolecule. To overcome these limitations, scientists can turn to another set of approaches, single-molecule biophysics. In these methods, the ensemble reaction is reduced to a smaller number of interacting partners (e.g., a single DNA interacting with one or multiple proteins) and a probe used to manipulate and/or observe the process. The goal of this Alert equipment bid is to apply such approaches to fundamental biological problems by bringing a cutting-edge single-molecule microscope to the Wolfson Bioimaging Facility (WBF) at the University of Bristol.

The instrument we want to fund is called a C-trap - a combined optical tweezers and confocal fluorescence microscope. An optical tweezers uses a focussed laser beam to trap a small particle, typically an ~1 micron latex bead. Photons from the laser have momentum, and refraction or reflection caused by the bead changes their path hence changing their momentum. By conservation of energy, equal and opposite forces are produced on the bead, trapping the particle. Accordingly, moving the laser focus will result in corresponding motion of the bead in 3D. The C-trap can produce up to four of these traps simultaneously. By attaching biomolecules to the beads, we can not only move them at will, we can also measure forces acting on them from the displacement of the trapped bead. For example, a molecule of DNA can be tethered between two beads and stretched into different conformations. To observe single proteins interacting with the DNA, the C-trap integrates confocal scanning lasers that can excite fluorescent molecules using up to 3 colours. We can then create movies of 3 different proteins moving and interacting on a single DNA molecule - we term this "visual biochemistry". Synchronisation and disorder are no longer limitations as the individual reactions can be compared. The C-trap is a very sophisticated instrument for correlating force measurements with accurate fluorescent positioning, with minimal user-intervention. Its relative ease-of use makes it an ideal instrument for a multi-user facility such as the WBF.

To establish the technique in the WBF, the C-trap will be first used by a group of labs who study protein interactions with DNA and RNA. For the first time they will be able to directly observe: how genes are expressed (transcription); how DNA damage is dealt with (DNA repair); how chromosomes are packaged during cell division and how that packaging influences access by other proteins; the production of proteins from RNA (translation); and how enzymes like CRISPR-Cas recognise specific DNA sequences during gene editing. The team will be assisted in microscope operation and in the analysis of data by a research technical professional, Stephen Cross, a key member of the WBF with biophysics training. He will ensure that we get the most benefit from the instrument. He will also promote the use of the instrument to a wider group of users once protocols for use are established. A particularly important goal is to bolster the employability of the younger members of our teams by training them in interdisciplinary science.

Single-molecule techniques are a powerful way to decipher complex molecular interactions, by allowing direct, real-time observation and measurement of parameters such as force that are challenging in ensemble reactions. Here, we want to establish a combined optical tweezers and confocal scanning fluorescence microscope (Lumicks C-trap G2) as the first multi-user single-molecule biophysics instrument in the Wolfson BioImaging Facility (WBF) at the University of Bristol. The flagship WBF provides microscope access and training for >400 investigators from Bristol and wider scientific communities. The C-trap combines turnkey operation, ideal for a facility, with state-of-the-art specifications; the system will have up to 4 low-drift optical traps capable of high-resolution force measurements, correlated with 3-colour scanning confocal imaging with single photon sensitivity. The instrument will be used first by 7 groups studying fundamental mechanisms of DNA repair and packaging, gene expression control during transcription and translation, and target-site binding fidelity of gene editing enzymes. These projects require manipulation of polynucleotides using either 2-traps (e.g., DNA stretching) or 4-traps (e.g., DNA knotting). Simultaneously, we need to localise multiple labelled biomolecules as they undergo ordered assembly and motion on the polynucleotides, which will be possible due to the true correlation with confocal imaging. Central to the grant's success is a research technical professional in the WBF, Stephen Cross, who has a single-molecule biophysics background and extensive microscopy data analysis experience. He will help launch the first phase projects and, in the second phase, will help grow our userbase to other molecular scientists, including from the Bristol GW4 University Alliance (Exeter, Bath, Cardiff). A legacy of this instrument will be a cohort of early career researchers with expertise in molecular biophysics and data-intensive analysis.