DNA:RNA hybrid duplex plays important roles in various biological processes. Both its structural stability and interactions with proteins are highly sequence dependent. In this study, we utilize homebuilt optical tweezers to investigate how GC contents in the sequence influence the structural and mechanical properties of DNA:RNA hybrid by measuring its contour length, elasticities, and overstretching dynamics. Our results support that the DNA:RNA hybrid adopts a conformation between the A-and B-form helix, and the GC content does not affect its structural and elastic parameters obviously when varying from 40 to 60% before the overstretching transition of DNA:RNA hybrid occurs. In the overstretching transition, however, our study unravels significant heterogeneity and strong sequence dependence, suggesting the presence of a highly dynamic competition between the two processes, namely the S-form duplex formation (nonhysteretic) and the unpeeling (hysteretic). Analyzing the components left in DNA:RNA hybrid after the overstretching transition suggests that the RNA strand is more easily unpeeled than the DNA strand, whereas an increase in the GC content from 40 to 60% can significantly reduce unpeeling. Large hysteresis is observed between the stretching and relaxation processes, which is also quantitatively correlated with the percentage of unpeeling in the DNA:RNA duplex. Increasing in both the salt concentration and GC content can effectively reduce the hysteresis with the latter being more significant. Together, our study reveals that the mechanical properties of DNA:RNA hybrid duplexes are significantly different from double-stranded DNA and double-stranded RNA, and its overstretching behavior is highly sequence dependent. These results should be taken into account in the future studies on DNA:RNA-hybrid-related functional structures and motor proteins.

DNA–RNA hybrid (DRH) plays important roles in many biological processes. Here, we use a thermodynamic theory to analyze the free energy and unpeeling properties of the overstretching transition for the DRH molecule and compare the results with double-helix DNA. We report that the RNA strand of DRH is easier to get unpeeled than the DNA strand while the difficulty in unpeeling the double helix DNA lies in between. We also investigate the sequence effect, such as GC content and purine content, on the properties of unpeeling the DRH. Further, to study the temperature effect, the force-temperature phase diagram of DRH and DNA are calculated and compared. Finally, using a kinetic model, we calculate the force–extension curves in the DRH stretching and relaxation process under different pulling rates and temperatures. Our results show that both pulling rate and temperature have important influences on the stretching and relaxation kinetics of unpeeling the DRH. Putting all these results together, our work provides a comprehensive view of both the thermodynamics and kinetics in DRH overstretching.

Trapping and manipulating micro-objects and high precision measurement of tiny force and displacements are of significance in physical and biological studies. Conventional optical tweezers rely on a tightly focused beam formed by a bulky microscope system. Currently, flat lenses, especially metalenses, have become emerging platforms for miniature optical tweezers application. Compared to traditional objectives, metalenses can be integrated into the sample chamber, so as to realize chip-scale light manipulation. Here, a transmissive and polarization-insensitive water-immersion metalens constructed by adaptive nano-antennas is experimentally proposed with an ultra-high numerical aperture (1.28) and a high focusing efficiency (~ 50%) at the wavelength of 532 nm. With it, a stable optical trapping has been demonstrated with the lateral trapping stiffness of more than 500 pN/(µm·W), which reaches the same order of magnitude as a conventional objective and shows better performance than other reported flat lenses. In addition, bead steering experiment exhibits lateral manipulation range more than 2 µm, including the region of approximately 0.5 µm with little changes in stiffness. We believe that this metalens enables chip-scale optical tweezers, making optical trapping and manipulating easy, reliable, high-performance and more compatible with prevalent optical tweezers applications such as single-molecule or single-cell experiments.

Single-molecule force spectroscopy (SMFS) measurements of the dynamics of biomolecules typically require identifying massive events and states from large data sets, such as extracting rupture forces from force-extension curves (FECs) in pulling experiments and identifying states from extension-time trajectories (ETTs) in force-clamp experiments. The former is often accomplished manually and hence is time-consuming and laborious while the latter is always impeded by the presence of base-line drift. In this study, we attempt to accurately and automatically identify the events and states from SMFS experiments with a machine learning approach, ACCESS (approach combining clustering and classification for event identification of SMFS). As demonstrated by analysis of a series of data sets, ACCESS can extract the rupture forces from FECs containing multiple unfolding steps and classify the rupture forces into the corresponding conformational transitions. Moreover, ACCESS successfully identifies the unfolded and folded states even though the ETTs display severe non-monotonic base-line drift. Besides, ACCESS is straightforward in use as it requires only three easy-to-interpret parameters. As such, we anticipate that ACCESS will be a useful, easy-to-implement and high-performance tool for event and state identification across a range of single-molecule experiments.