Article
Biochemistry & Molecular Biology
Javier Jarillo, Borja Ibarra, Francisco Javier Cao-Garcia
Summary: The analysis and modeling techniques of in vitro single-molecule DNA replication events play a crucial role in understanding the process, with a focus on the real-time kinetics of DNA polymerase and DNA helicase. Proper analysis of single-molecule data is essential for obtaining a detailed picture of the system's dynamics, including kinetics rates, equilibrium constants, and conformational changes. These techniques can also be adapted to study the operation of other proteins involved in nucleic acids metabolism.
COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL
(2021)
Article
Chemistry, Multidisciplinary
Marta Urbanska, Annemarie Ludecke, Wilhelm J. Walter, Antoine M. van Oijen, Karl E. Duderstadt, Stefan Diez
Summary: This study introduces a highly-parallel, microfluidics-based method for rapid collection of force-dependent motility parameters of cytoskeletal motors, significantly improving throughput. DNA-tethered beads are used to apply tunable hydrodynamic forces to stepping kinesin-1 motors, allowing for simultaneous tracking of various motility parameters of hundreds of individual molecules. The approach, applicable to other molecular systems, represents a new methodology for parallelized single-molecule force studies on cytoskeletal motors.
Article
Chemistry, Analytical
Diana Soukarie, Philippe Rousseau, Maya Salhi, Alexia de Caro, Jean-Marc Escudier, Catherine Tardin, Vincent Ecochard, Laurence Salome
Summary: This study successfully developed an aptasensor based on high-throughput single-molecule techniques, which enables rapid, simple, and sensitive detection of single target molecules. The presence and concentration of the target molecules can be monitored in real time by combining videomicroscopy and image analysis.
ANALYTICAL CHEMISTRY
(2022)
Article
Multidisciplinary Sciences
Kacey N. Mersch, Joshua E. Sokoloski, Binh Nguyen, Roberto Galletto, Timothy M. Lohman
Summary: RPA is an essential single-stranded DNA-binding protein that can bind and diffuse along single-stranded DNA. It has been shown that Pif1 can use its ATP-dependent translocase activity to push RPA along single-stranded DNA. Furthermore, Pif1 can also push RPA from single-stranded DNA into double-stranded DNA, resulting in stable disruption of at least 9 base pairs of duplex DNA. These findings highlight the dynamic nature of RPA and provide insights into the mechanism of directional DNA unwinding.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2023)
Review
Biochemistry & Molecular Biology
Matthew A. Schaich, Bennett Van Houten
Summary: DNA is constantly threatened by various chemical and physical insults, requiring multiple repair pathways to maintain genome stability. Repair proteins' mechanisms of detecting damaged substrates in cellular chromatin remain unclear, but recent single-molecule techniques allow for visualization of damage search and detection processes.
FRONTIERS IN MOLECULAR BIOSCIENCES
(2021)
Review
Chemistry, Multidisciplinary
Yanfang Wu, J. Justin Gooding
Summary: This review provides an overview of the concepts, fabrication, and applications of nanopore sensors, with a focus on their potential and recent developments in quantitative analysis.
CHEMICAL SOCIETY REVIEWS
(2022)
Article
Chemistry, Multidisciplinary
Aleksandra K. Adamczyk, Teun A. P. M. Huijben, Miguel Sison, Andrea Di Luca, Stefano Vanni, Sophie Brasselet, Kim I. Mortensen, Fernando D. Stefani, Mauricio Pilo-Pais, Guillermo P. Acuna
Summary: This study demonstrates a controlled orientation of single molecules in DNA origami by linking them to oligonucleotide strands and leaving unpaired bases. The number of unpaired bases affects the stretching and orientation of the fluorophore linkers. These findings expand the application of DNA origami in the fabrication of nanodevices involving orientation-dependent molecular interactions.
Article
Optics
Wenqi Zhao, Xiaochaoran Tian, Zhening Fang, Shiyi Xiao, Meng Qiu, Qiong He, Wei Feng, Fuyou Li, Yuanbo Zhang, Lei Zhou, Yan-Wen Tan
Summary: Single-molecule fluorescence detection is a powerful tool for studying molecular dynamics in bioscience, with near-infrared (NIR) fluorescence offering advantages such as higher signal-to-noise ratio and increased tissue penetration depth. However, the low quantum yield of most NIR fluorophores poses challenges for single-molecule detection. This study used asymmetric plasmonic nano-antennas to enhance the fluorescence intensity of a typical NIR dye by up to 405 times, primarily due to increasing the quantum yield. Coupled-mode-theory analysis revealed that the enhancements stemmed from resonance-matching between antenna and molecule, as well as optimizing the coupling between near- and far-field modes with designer asymmetric structures. This work provides a universal scheme for engineering single-molecule fluorescence in the near-infrared regime.
LIGHT-SCIENCE & APPLICATIONS
(2021)
Review
Biochemistry & Molecular Biology
Shunsuke Takahashi, Masahiko Oshige, Shinji Katsura
Summary: DNA replication, repair, and recombination in cells regulate genetic information transfer. Single-molecule imaging provides new insights into the dynamic behaviors of individual biomolecules, helping analyze the interaction between DNA and proteins.
Article
Biochemistry & Molecular Biology
Joseph Tibbs, Mohamed Ghoneim, Colleen C. Caldwell, Troy Buzynski, Wayne Bowie, Elizabeth M. Boehm, M. Todd Washington, S. M. Ali Tabei, Maria Spies
Summary: Molecular machines within cells exhibit dynamic assembly, disassembly, and reorganization, with molecular interactions between components being observed and quantified through single-molecule level studies and fluorescence microscopy techniques. Analyzing sequences of molecular interactions can reveal the structure and dynamic organization of complexes, providing important insights into complex biological systems.
NUCLEIC ACIDS RESEARCH
(2021)
Article
Multidisciplinary Sciences
Meenakshi Sharma, Eva de Alba
Summary: This study investigates the association and dissociation rates of the AIM2-DNA complex at the single molecule level using optical traps and confocal fluorescence microscopy. The results reveal distinct mechanisms for the growth of AIM2 oligomers and show that the size of the oligomers can increase fourfold within seconds. Additionally, it is found that single AIM2 molecules do not diffuse/scan along the DNA, suggesting that oligomerization depends on stochastic encounters with DNA and/or DNA-bound AIM2.
NATURE COMMUNICATIONS
(2023)
Review
Biochemistry & Molecular Biology
Longfu Xu, Matthew T. J. Halma, Gijs J. L. Wuite
Summary: Single-stranded DNA-binding proteins (SSBs) are essential for DNA metabolism and play crucial roles in maintaining genome integrity and coordinating with other proteins involved in DNA replication, recombination, and repair. Recent advances in single-molecule techniques and structural methods have greatly improved our understanding of the binding dynamics and interaction of SSBs with ssDNA and other protein partners. We highlight the central coordination role of SSBs in directly modulating the activities of other proteins and discuss the various modes of interaction between SSBs and their protein partners, providing a comprehensive view of the interaction network shaped by SSBs.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Chemistry, Multidisciplinary
Lars Richter, Alan M. Szalai, C. Lorena Manzanares-Palenzuela, Izabela Kaminska, Philip Tinnefeld
Summary: The study explores the use of fluorescence-based single-molecule techniques in the research of 2D materials and the application of DNA as an anchor for single dye molecules. It reviews the existing literature on the combination of these two fields and discusses the potential synergies that can be achieved.
ADVANCED MATERIALS
(2023)
Article
Physics, Fluids & Plasmas
Ting Liang, Chao Yang, Xiaoya Song, Yuyu Feng, Yanhui Liu, Hu Chen
Summary: This study systematically investigated the kinetics and thermodynamics of the folding-unfolding transition of an individual DNA hairpin in a crowded environment using a combination of high-resolution magnetic tweezers and an extended crowder-oxDNA model. The results demonstrated a linear dependence of the critical force of the DNA hairpin on the polyethylene glycol (PEG) concentration, highlighting the importance of excluded volume effects in the interaction between PEG and the DNA hairpin. The method provides insights into accurately mimicking cytosolic environments using mixtures of different inert molecules. Overall rating: 9/10.
Article
Chemistry, Multidisciplinary
Yufeng Pei, Tianyuan Bian, Yonglin Liu, Yan Liu, Yujie Xie, Jie Song
Summary: This study developed a real-time, label-free, and force-controlled single-molecule DNA computing method. By using stretching forces and tracking real-time signals, Boolean logic computing was achieved at the single-molecule level with output determined at a single base-pair resolution.
Article
Multidisciplinary Sciences
Aakash Basu, Dmitriy G. Bobrovnikov, Zan Qureshi, Tunc Kayikcioglu, Thuy T. M. Ngo, Anand Ranjan, Sebastian Eustermann, Basilio Cieza, Michael T. Morgan, Miroslav Hejna, H. Tomas Rube, Karl-Peter Hopfner, Cynthia Wolberger, Jun S. Song, Taekjip Ha
Summary: Mechanical deformations of DNA, such as bending, are common and have been implicated in various cellular functions. A high-throughput assay called 'loop-seq' was developed to measure DNA looping propensity and intrinsic cyclizabilities of DNA fragments across different genomic regions. The study found regions of low bendability in nucleosome-depleted regions, which affects nucleosome sliding and gene transcription. Overall, the study shows that local DNA mechanics have broad functional implications on chromatin organization and transcription.
Article
Multidisciplinary Sciences
Yanbo Wang, John Mallon, Haobo Wang, Digvijay Singh, Myung Hyun Jo, Boyang Hua, Scott Bailey, Taekjip Ha
Summary: The research discovered that the HNH domain of CRISPR-Cas9 exhibits high flexibility post-DNA cleavage, exposing a 3' flap after cleavage of the non-target strand, potentially interacting with the DNA repair machinery. These findings reveal the regulatory role of DNA cleavage activity on Cas9 conformation and suggest potential applications in biotechnology.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Chemistry, Multidisciplinary
Myung Hyun Jo, Byoung Choul Kim, Keewon Sung, Reynold A. Panettieri, Steven S. An, Jian Liu, Taekjip Ha
Summary: The study found that cell shortening and focal adhesion disassembly occurred after an increase in intracellular calcium levels induced by histamine. A mathematical model predicted that stabilizing focal adhesions hinders cell shortening, while focal adhesion disruption is preceded by strengthening due to actomyosin-activated tension.
Article
Multidisciplinary Sciences
Nilmani Singh, Adriana Reyes-Ordonez, Michael A. Compagnone, Jesus F. Moreno Castillo, Benjamin J. Leslie, Taekjip Ha, Jie Chen
Summary: This study explores the specific interactions of human PH domain-containing proteins with PIPs and reveals unexpected lipid-binding specificity among these proteins. By analyzing the structural requirements and functional relevance of PIP binding, as well as using a prediction algorithm, the researchers demonstrate widespread PIP binding among human PH domains.
NATURE COMMUNICATIONS
(2021)
Review
Biochemistry & Molecular Biology
Taekjip Ha, Christian Kaiser, Sua Myong, Bin Wu, Jie Xiao
Summary: Due to their unique abilities to manipulate, label, and image individual molecules, single-molecule techniques provide unprecedented access to elementary biological processes.
Article
Multidisciplinary Sciences
Li Dai, Digvijay Singh, Suoang Lu, Vishal Kottadiel, Reza Vafabakhsh, Marthandan Mahalingam, Yann R. Chemla, Taekjip Ha, Venigalla B. Rao
Summary: The study revealed that the active bacteriophage T4 DNA packaging motor is a pentamer and is able to tolerate inactive subunits. However, motors with inactive subunits showed decreased DNA engagements, higher failure rates in encapsidation, reduced packaging velocity, and increased pausing. This suggests that strict coordination among motor subunits is not crucial for the function of packaging motors.
NATURE COMMUNICATIONS
(2021)
Article
Chemistry, Multidisciplinary
Hossein Moghimianavval, Chintan Patel, Sonisilpa Mohapatra, Sung-Won Hwang, Tunc Kayikcioglu, Yashar Bashirzadeh, Allen P. Liu, Taekjip Ha
Summary: InterSpy is introduced as a synthetic biology tool for engineering membrane-membrane interfaces. It allows tracking and reconstitution of functional fluorescent protein between apposing synthetic or cell membranes, demonstrating the potential for designing non-native cellular communication pathways and creating synthetic tissues. The technology is tested using a mammalian cell-free expression system and shows promising results.
Article
Biology
Claudia C. Carcamo, Matthew F. Poyton, Anand Ranjan, Giho Park, Robert K. Louder, Xinyu A. Feng, Jee Min Kim, Thuc Dzu, Carl Wu, Taekjip Ha
Summary: This study characterizes the 1D scanning properties of SWR1, a chromatin remodeler, and reveals the impacts of various factors on its scanning process. By directly observing SWR1 diffusion on DNA, the study demonstrates that ATP and a DNA-binding subunit play important roles in the overall diffusive behavior of the complex. The findings provide valuable insights into the role of SWR1 in chromatin remodeling.
Article
Cell Biology
Roger S. Zou, Alberto Marin-Gonzalez, Yang Liu, Hans B. Liu, Leo Shen, Rachel K. Dveirin, Jay X. J. Luo, Reza Kalhor, Taekjip Ha
Summary: Here, we propose an approach that combines CRISPR system with high-throughput sequencing to target hundreds of epigenetically diverse endogenous genomic sites simultaneously, measuring Cas9 dynamics and cellular responses. The use of multi-target guide RNAs (mgRNAs) enables massive multiplexing of CRISPR, providing insights into Cas9 binding, cleavage, and cellular response to DNA damage.
NATURE CELL BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Momcilo Gavrilov, Joshua Y. C. Yang, Roger S. Zou, Wen Ma, Chun-Ying Lee, Sonisilpa Mohapatra, Jimin Kang, Ting-Wei Liao, Sua Myong, Taekjip Ha
Summary: Polymerase Chain Reaction (PCR) is a crucial method in molecular diagnostics and life sciences. This study introduces a new method, SHARP (SSB-Helicase Assisted Rapid PCR), which uses an engineered helicase and single-stranded DNA binding protein (SSB) to replace the heating step of PCR. The new method allows the isothermal amplification of DNA fragments up to 6000 base pairs.
NATURE COMMUNICATIONS
(2022)
Article
Chemistry, Physical
Ikenna C. Okafor, Taekjip Ha
Summary: CRISPR Cas9 is an RNA-guided endonuclease that is a part of bacterial adaptive immune system. By developing a single molecule FRET assay, the study investigated the conformational changes of sgRNA and the binding of Cas9 to sgRNA, providing insights into the assembly dynamics of Cas9 RNA ribonucleoprotein complex. This research could contribute to the rational design of sgRNAs and improve the editing outcomes.
JOURNAL OF PHYSICAL CHEMISTRY B
(2023)
Article
Biochemistry & Molecular Biology
Aakash Basu, Dmitriy G. Bobrovnikov, Basilio Cieza, Juan Pablo Arcon, Zan Qureshi, Modesto Orozco, Taekjip Ha
Summary: In this study, we comprehensively characterized the mechanical code of DNA using high-throughput experimental methods and developed a physical model to describe the sequence and methylation dependence of DNA deformation. Our measurements and model validations demonstrated that sequence and epigenetic modifications can encode regulatory information in diverse contexts.
NATURE STRUCTURAL & MOLECULAR BIOLOGY
(2022)
Article
Multidisciplinary Sciences
Yanbo Wang, W. Taylor Cottle, Haobo Wang, Momcilo Gavrilov, Roger S. Zou, Minh-Tam Pham, Srinivasan Yegnasubramanian, Scott Bailey, Taekjip Ha
Summary: We describe a method called sgGOLDFISH for direct visualization of point mutations in situ, which can differentiate wild-type and mutant sequences with single-nucleotide sensitivity. This method has potential applications in studying genetic diseases and nuclear biology.
NATURE COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Aman Y. Husbands, Antje Feller, Vasudha Aggarwal, Courtney E. Dresden, Ashton S. Holub, Taekjip Ha, Marja C. P. Timmermans
Summary: The START domain of the HD-ZIPIII transcription factors plays a crucial role in promoting homodimerization and increasing transcriptional potency, while also binding to phospholipids. This discovery resolves a long-standing mystery in plant development and highlights the regulatory potential of this evolutionary module.