Article
Chemistry, Multidisciplinary
Sung Hyun Kim, Hyunwoo Kim, Hawoong Jeong, Tae-Young Yoon
Summary: The study demonstrates the use of single-molecule FRET technology to encode virtual signals in DNA barcodes, allowing for precise measurement of FRET efficiency for each binding event and differentiation of six DNA barcodes.
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
Biology
Sarah R. Hansen, David S. White, Mark Scalf, Ivan R. Correa, Lloyd M. Smith, Aaron A. Hoskins, Jonathan P. Staley
Summary: This study investigated the pathway of 5' SS selection by purified yeast U1 snRNP using colocalization single-molecule spectroscopy. The results revealed a sequence-dependent, two-step mechanism for U1 to reversibly select 5' SS, providing a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS.
Article
Chemistry, Multidisciplinary
Anoja Megalathan, Kalani M. Wijesinghe, Soma Dhakal
Summary: The article presents a background-free ultrasensitive detection sensor on a simple platform for p53 tumor suppressor gene, showing continuous dynamic switching between low- and high-FRET states in the presence of a target and detecting down to low femtomolar (fM) concentration. The sensor is also highly effective in discriminating against single-nucleotide polymorphisms (SNPs), offering a wide range of nucleic acid sequences detection for early disease diagnosis and genetic disorder screening.
Article
Multidisciplinary Sciences
Yanyan Xue, Jun Li, Dian Chen, Xizhu Zhao, Liang Hong, Yu Liu
Summary: This study develops a method to monitor the conformational change of riboSAM during transcription, and observes five co-existing states in riboSAM. A bifurcated helix structure is identified in one of the states and is found to be responsible for translation inhibition. This strategy enables the precise mapping of RNA conformational landscape during transcription.
NATURE COMMUNICATIONS
(2023)
Article
Multidisciplinary Sciences
Adrien Chauvier, Patrick St-Pierre, Jean-Francois Nadon, Elsa D. M. Hien, Cibran Perez-Gonzalez, Sebastien H. Eschbach, Anne-Marie Lamontagne, J. Carlos Penedo, Daniel A. Lafontaine
Summary: The researchers presented a new approach for site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes, revealing the relationship between RNA folding and gene regulation.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Biochemistry & Molecular Biology
Christin Fuks, Sebastian Falkner, Nadine Schwierz, Martin Hengesbach
Summary: Riboswitch RNAs regulate gene expression through conformational changes induced by environmental conditions and specific ligand binding, which is of great significance for understanding the mechanism of RNA regulation.
FRONTIERS IN MOLECULAR BIOSCIENCES
(2022)
Article
Optics
Raymundo Moya, Toru Kondo, Audrey C. Norris, Gabriela S. Schlau-Cohen
Summary: Single-molecule spectroscopy is widely used to study heterogeneity in behaviors on different timescales, with the recent emergence of single-molecule pump-probe spectroscopy for detecting heterogeneity on femtosecond and picosecond timescales. By developing a tunable apparatus, a bimodal distribution of energetic relaxation time constants was found for the fluorescent dye Atto647N, indicating dominance of specific processes within individual molecules and low heterogeneity. This spectrally-tunable single-molecule pump-probe spectroscopy has the potential to investigate heterogeneity in various biological and material systems.
Article
Nanoscience & Nanotechnology
Satyajit Patra, Jean-Benoit Claude, Jerome Wenger
Summary: This study thoroughly investigates the influence of zero-mode waveguide (ZMW) on the fluorescence of single molecules. The results show that the photostability is preserved in ZMW and both fluorescence brightness and total number of emitted photons are increased. Additionally, it is demonstrated that nanophotonics can enhance the FRET process.
Review
Biochemistry & Molecular Biology
Mohamed A. Sobhy, Muhammad Tehseen, Masateru Takahashi, Amer Bralic, Alfredo De Biasio, Samir M. Hamdan
Summary: FEN1 is a key component in the molecular machinery of DNA replication and repair. The study reviewed in this article focuses on using single-molecule fluorescence to study important mechanistic details of FEN1 enzymatic reaction, providing a guide for studying other biomolecular enzymatic reactions. The research shows that FEN1 actively bends the substrate to control active site assembly, preventing off-target cleavage.
COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL
(2021)
Article
Biophysics
Tapas Paul, Taekjip Ha, Sua Myong
Summary: This study presents strategies for regenerating PEG surfaces for multiple rounds of experiments, including washing out bound proteins, reannealing double-stranded DNA, and stripping off NeutrAvidin to regenerate the biotin-PEG layer. These methods enhance the efficiency of single-molecule experiments.
BIOPHYSICAL JOURNAL
(2021)
Article
Chemistry, Physical
Cecilia Zaza, German Chiarelli, Ludovit P. Zweifel, Mauricio Pilo-Pais, Evangelos Sisamakis, Fabio Barachati, Fernando D. Stefani, Guillermo P. Acuna
Summary: Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
Article
Chemistry, Multidisciplinary
Iwo Konig, Andrea Soranno, Daniel Nettels, Benjamin Schuler
Summary: The concentrations of macromolecules within cells can affect the conformations and dynamics of proteins. By reducing cell volume through hyperosmotic stress, it was found that intrinsically disordered proteins exhibit compaction and slower dynamics in more crowded cells. This concentration and length-scale dependence of crowding can be explained by polymer theory and depletion interactions.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2021)
Article
Biochemical Research Methods
Jack W. Shepherd, Sarah Lecinski, Jasmine Wragg, Sviatlana Shashkova, Chris MacDonald, Mark C. Leake
Summary: This study used cutting-edge genomically integrated FRET biosensing to determine localized molecular crowding in single live yeast cells, finding that crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicated that the cell membrane is largely inaccessible to these sensors, and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds.
Article
Multidisciplinary Sciences
David Scheerer, Bharat V. Adkar, Sanchari Bhattacharyya, Dorit Levy, Marija Iljina, Inbal Riven, Orly Dym, Gilad Haran, Eugene I. Shakhnovich
Summary: Enzymes have a crucial role in controlling chemical reactions and achieving high catalytic efficiency and selectivity. The three-domain enzyme adenylate kinase (AK) catalyzes phosphotransfer between ATP and AMP. Our study using single-molecule FRET spectroscopy reveals that AMP inhibits AK by promoting faster and more cooperative domain closure by ATP, leading to an increased population of the closed state. We developed a model based on experimentally observed opening and closing rates, explaining the complex activity of AK, particularly substrate inhibition, and highlighting the importance of protein motions in enzymatic activity.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2023)
Article
Biochemical Research Methods
K. Ramki, G. Thiruppathi, Selva Kumar Ramasamy, P. Sundararaj, P. Sakthivel
Summary: A chromone-based ratiometric fluorescent probe L2 was developed for the selective detection of Hg(II) in a semiaqueous solution. The probe exhibited enhanced fluorescence in its aggregated state and even higher fluorescence when chelated with Hg(II). The probe demonstrated high sensitivity and specificity for Hg(II) detection and was successfully applied for imaging Hg(II) in a living model.
Article
Biochemical Research Methods
Qun Zhang, Rui Yang, Gang Liu, Shiyan Jiang, Jiarui Wang, Juqiang Lin, Tingyin Wang, Jing Wang, Zufang Huang
Summary: This research aims to develop a cost-effective and portable method for measuring creatinine levels using the enhanced Tyndall effect phenomenon. The method offers a promising solution for monitoring renal healthcare in resource-limited settings.
