All aspects of human health, from gene regulation in cells to the efficacy of drugs are governed by the interactions of individual molecules. Advancing human health thus depends critically on our ability to observe and interpret the finest details of these interactions
All aspects of human health, from gene regulation in cells to the efficacy of drugs are governed by the interactions of individual molecules. Advancing human health thus depends critically on our ability to observe and interpret the finest details of these interactions. Conventional tools typically interrogate millions of molecules at a time and report one aggregate signal in “ensemble” measurements. Because these can miss rare events, and obscure the heterogeneous and dynamic nature of biomolecules, they have limited ability to help us understand the individual reactions that cause a gene to become improperly expressed or a drug to fail.
Yet imaging single molecules is difficult: molecules bounce around rapidly at the micron scale and leave the field of view of a powerful microscope in less than one-thousandth of a second. Prof. Sabrina Leslie and her team at McGill University have pioneered a single-molecule microscopy technique called “CLiC” (Convex Lens-induced Confinement).
CLiC: Convex Lens-induced Confinement
CLiC makes it possible to watch naturally fluctuating biomolecules as they diffuse and interact, as they search for and bind to each other. It works by squeezing individual molecules into an array of open-face, nanoscale features in a transparent surface. This allows many molecules to be observed at once, with single-molecule resolution, for long observation periods, and under a large range of “cell-like” conditions including high concentrations and in the presence of complex solutions. The imposed “entropic” confinement enables reagents to be exchanged without losing sight of the molecules so that their complete response can be imaged sequentially and in real time.
Prof. Leslie and her team have recently published two articles which apply CLiC to deconstruct two kinds of molecular interactions in “cell-like conditions” – nucleic acid interactions and protein interactions.
DNA supercoiling
You’re likely familiar with the famous shape of DNA - the double helix. But DNA forms complex structures at many levels above the double helix, and understanding how these structures form and impact the functioning of DNA in health and disease is an active area of scientific research. In this article, Prof. Leslie’s team in collaboration with Prof. Levens at the National Cancer Institute and Prof. Benham at the UC Davis Genome Center, used the single-molecule “CLiC” microscopy technique to investigate so-called DNA supercoiling. DNA supercoiling occurs when DNA is over- or underwound, like twisting an old telephone cord, and results from both the packing of DNA into chromosomes and the action of other molecules. This article investigates how the extent of supercoiling can impact the tendency of a DNA molecule to open up and become accessible to the complex molecular machinery that latches onto and reads the DNA molecule to generate the vast library of proteins that build and run our bodies. These insights are important to developing therapies that involve designing molecules to target or edit specific sites, in addition to developing cancer diagnostics that are sensitive to the structural state of DNA.
This publication studies how “cell-like conditions” like crowding agents and ions in solution impact the dynamics and interactions of nucleic acids. The fundamental insights from this work are applicable to the development of a wide range of nucleic acid based therapeutics (such as antisense oligonucleotides) and biotechnologies (such as CRISPR-Cas9 gene editing), whose success relies upon understanding the interactions between molecules.
For more, read the article published in the journal Nucleic Acids Research
Organizing molecules
The cells in our bodies contain many different types of molecules, which begs the question of how to organize them efficiently for everything to function properly. A particularly interesting organizational strategy is the dynamic formation and disassembly of the liquid-like NMOs. A challenge in studying NMOs has been that model droplets formed outside of a cell are much larger (like oil droplets in water that fuse together) than those formed inside a cell, and it is unclear how this size difference might affect droplet properties and behaviour. Prof. Leslie’s lab in collaboration with Prof. Stephen Michnick’s lab in the Université de Montreal Biochemistry Department, has applied the single-molecule “CLiC” technique to control the sizes of droplets, watch them form, and measure their internal properties. As recently described in the Journal of the American Chemical Society, these scientists have examined two different biopolymer NMO systems and discovered that droplets can have size-dependent properties. Using CLiC imaging, the team monitored the formation and disassembly of droplets in real time, and was able to access the nano and micro scale dimensions relevant to cellular environments. The ability to image these complex dynamics on nanoscale dimensions, with complete control over the biophysical and chemical environment, provides an important platform for investigating a wide range of neurodegenerative diseases known to be associated with structural and organizational properties of the proteins.
This work opens up new possibilities for studies of “non membranous organelles” (NMOs), small droplet-like structures which have been discovered inside cells and which seem to spatially organize molecules like proteins. This work introduces direct visualization of NMOs under confined conditions that emulate live cells, with simplicity and control over the sample. Since problems with NMO formation and disassembly are associated with cancer, Alzheimer’s, and Parkinson’s Diseases, among others, a better understanding of these interesting and complex structures could greatly impact health research.
To read the full article: Journal of the American Chemical Society
For more about Sabrina Leslie
Contact:
Cynthia Lee
McGill Media Relations Office
514-398-6754