BMB Reports 2025; 58(1): 1-1  https://doi.org/10.5483/BMBRep.2025-0005
Attacking biological problems through single-molecule approaches
Jong-Bong Lee
Department of Physics, Division of Interdisciplinary Bioscience & Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea
Correspondence to: E-mail: jblee@postech.ac.kr
Received: December 30, 2024; Revised: December 30, 2024; Accepted: January 31, 2025; Published online: January 31, 2025.
© Korean Society for Biochemistry and Molecular Biology. All rights reserved.

cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In the last few decades, single-molecule techniques have emerged as transformative tools for exploring biological problems. By observing and analyzing individual molecules, these methods make it possible to investigate fundamental dynamics of biomolecular processes deeper. Unlike traditional ensemble methods that average the behavior of populations, single-molecule approaches provide a unique window to observe molecular heterogeneity, transient interactions, and dynamic processes that are otherwise hidden. This special issue brings together six mini-reviews that present how these cutting-edge methodologies are advancing our understanding of diverse and complex biological systems. Each review highlights unique applications, significant breakthroughs, and ongoing challenges. They collectively demonstrate the versatility and impact of single-molecule techniques.
Keywords: BMB Reports Special Issue, single-molecule
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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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