
Phil Sang Yu et al. emphasize advancements in cryogenic single-molecule fluorescence imaging. Cryo-fixation can preserve biological samples in a near-native state and enhance photophysical properties of fluorophores, thus increasing photo-stability. They address key challenges in cryogenic imaging, including poor signal-to-noise ratios and limited numerical aperture in optical systems. Additionally, they discuss recent innovations such as the integration of super-resolution imaging techniques and the application of AI-driven image analysis to push the boundaries of the field.
Jeongmin Lee et al. explore CRISPR/Cas systems through single-molecule methods, uncovering mechanisms of target search, recognition, and cleavage. Their review examined dynamic behaviors of Cas proteins and their interactions with DNA, including PAM site recognition and R-loop formation critical for gene editing specificity and efficiency. Their insights underscore the immense potential of single-molecule approaches in advancing genome editing and biotechnological applications.
Donghun Lee et al. demonstrate the role of single-molecule techniques in elucidating the molecular mechanisms of base excision repair (BER), highlighting dynamic processes of lesion recognition, excision, and repair synthesis. They emphasize coordinated actions of key enzymes such as DNA glycosylases, APE1, and DNA polymerase β in maintaining genomic stability. Furthermore, they explore an emerging role of UV-damaged DNA binding protein in BER. These findings underline the power of single-molecule studies in uncovering the complexity of DNA repair pathways.
Sihyeong Nho et al. focus on nucleosome and chromatin fiber dynamics that are fundamental to gene regulation and chromosomal organization. They describe how single-molecule approaches have unveiled wrapping–unwrapping and condensation–decondensation dynamics influenced by ionic conditions, histone modifications, and DNA sequence context. These insights have profound implications for understanding roles of chromatin in gene expression and genome organization.
Jaehyeon Shin et al. review the integration of single-molecule techniques with model membrane systems, which mimic biological membranes. Innovations in lipid bilayers, nanodiscs, and vesicles have advanced fluorescence microscopy and force spectroscopy, uncovering mechanisms of ion channel function and membrane fusion. They emphasize how synergy between single-molecule approaches and model membranes is driving discoveries in membrane biology.
Sadaf Shehzad et al. introduce single-molecule DNA-flow stretching assays as versatile tools for studying DNA-protein interactions. They describe how these hybrid techniques can combine mechanical forces with fluorescence imaging to unravel mechanisms of DNA-binding proteins. Variants such as DNA curtain assays, DNA motion capture assays, and protein-induced fluorescence enhancement (PIFE) are highlighted for their multiplexed and robust capabilities. These innovative techniques will continue to expand our understanding of DNA-protein dynamics.
Collectively, these reviews bring attention to the groundbreaking power of single-molecule techniques to study a variety of biological problems. These approaches continue to push boundaries of biological research. With advancements of innovations in instrumentation, computational analysis, and experimental design, single-molecule methodologies will undoubtedly play a central role in answering next generation of biological questions. Together, these articles inspire continued exploration of molecular mechanisms that define life at its most fundamental level.
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