Optical tweezers are a high-resolution force measuring technique invented by A. Ashkin and colleagues in 1986. Optical tweezers, in brief, use a highly focused laser beam that can form a stable three-dimensional trap to manipulate micron-sized particles. Optical tweezers have sub-piconewton force resolution and sub-millisecond time response, which can be widely used in single-molecule biophysics. In single-molecule optical tweezers experiment, traditional optical tweezers geometries include single-trap and dual-trap geometries. Compared with the single-trap geometries, the dual-trap “dumbbell” assay has better stability and noise resistance, resulting in higher resolution and playing an important role in the study of DNA-protein interactions, protein folding and the mechanochemical properties of molecular motors. In this review, we provide an overview on the basic principles of optical tweezers and the experimental setup of the dual-beam optical tweezers in the National Facility for Protein Science in Shanghai. The application and progress of dual-beam optical tweezers in single-molecule biology are summarized, and we focus on investigating some perspectives for future applications.
When a photon is absorbed by an absorptive particle, the partial momentum of the photons is transferred to the particle, which in turn generates an optical trapping force that stabilizes the particle. Quantitative calculation of the optical trapping force depends on the wavelength of the trapping light and the size of the trapped particle. When the particle radius is close to or greater than the light wavelength, the optical trapping forces can be calculated from “ray-optics” model. When the particle size is smaller than the wavelength, the electromagnetic scattering theory is often chosen as the calculation model (Fig. 1).
Optical tweezers are mainly used in the study of single biomolecules such as proteins and nucleic acids. The systems commonly used in single-molecule optical tweezers experiments include single-trap optical tweezers, dual-trap optical tweezers and angular optical tweezers. These experimental systems involve a variety of geometries, which can be used to directly manipulate single molecules and measure mechanical relevant parameters (Fig. 2). Among them, the “dumbbell” geometry of the tweezers has better stability and noise immunity and higher resolution than other optical tweezers configurations, and these advantages make the tweezers widely applied in single-molecule mechanical properties. The National Facility for Protein Science in Shanghai developed high-precision dual-trap optical tweezers and successfully used them to study the folding dynamics of protein complexes (Fig. 3). In this system, in order to accurately obtain the important parameter of optical trapping force, we chose to use the power spectral density method to calibrate the optical trap stiffness (Fig. 4), and realized base-pair resolution on the measurement of tether extension on the dual-trap optical tweezers (Fig. 5).
Optical tweezers provide powerful single-molecule evidence to study the mechanical behavior of nucleic acid and proteins that constitute the major roles in the interpretation of the central dogma in molecular biology (Fig. 6). Stretching dsDNA with dual-trap optical tweezers helps us understand the elastic model of DNA and lays a foundation for exploring protein folding, DNA-protein complex interactions and mechanochemical properties of molecular motors. Dual-trap optical tweezers can reveal the protein folding process at the single-molecule level, detect subtle protein misfolding information, and measure the translation, folding and molecular regulation processes of multi-domain proteins in real time. All those studies offer single-molecule information for understanding and treating neurodegenerative diseases (Fig. 7). DNA-protein binding is closely related to the molecular mechanisms of DNA replication, repair and transcription. The ability of dual-trap optical tweezers to monitor DNA-protein interactions in real time at the single-molecule level has advanced the development of related molecular mechanisms and molecular dynamics (Fig. 8). In addition, dual-trap optical tweezers can be used to study the motion characteristics of molecular motors. Mechanochemical properties of molecular motors are understood by measuring parameters such as step size, velocity, and run length (Fig. 9). Dual-trap optical tweezers can also be used to reveal how molecular chaperones regulate the folding and assembly process of protein complexes to clarify the folding mechanism, and provide the single-molecule basis for physiological processes (Fig. 10).
In recent years, dual-trap optical tweezers have been developing continuously. Laser Raman spectroscopy tweezers (LRST) have enabled the simultaneous combination of single-molecule manipulation and Raman spectroscopy measurements without direct contact with the sample. Dual-trap Raman tweezers built on this basis can detect the interaction between cells or stretch a single cell to study the changes caused by deformation (Fig. 11). The combination of optical tweezers and single-molecule fluorescence detection breaks the limitation that optical tweezers can only measure in one-dimensional direction, which enables the study of complex conformational changes at three-dimensional level (Fig. 12). Building on this, the combination of ultra-high resolution imaging technology with dual-trap optical tweezers makes it possible to capture the dynamics of a single protein at high protein concentrations (Fig. 13). Besides, nano-optical tweezers are capable of ultra-precise localization of single nano-objects and can track the changing state of biological macromolecules at high resolution over long periods (Fig. 14).
After more than three decades of development, dual-trap optical tweezers have gradually formed a well-established experimental system in biological research, and the increased temporal and spatial resolution has further extended the application range of dual-trap optical tweezers. At the same time, dual-trap optical tweezers face many challenges in the development and biological application, for example, low throughput or low trap depth and efficiency of living cells. In recent years, although several commercial optical tweezers instruments have been launched for single-molecule studies to promote the single-molecule science, the use of optical tweezers to study single molecules is still developing in China. The National Facility for Protein Science in Shanghai is among the few labs to develop high-precision dual-trap optical tweezers for single-molecule studies. The instrument has high stability and a high signal-to-noise ratio, which has been used in biological single-molecule researches. It is expected that single-molecule experiment using optical tweezers would enter a new phase in China in the coming decade.
