Single-Molecule Dynamic Structural Biology with Vertically Arranged DNA on a Fluorescence Microscope

Chemistry Nanoscience Life Sciences Biochemistry Biophysics
single-molecule fluorescence DNA-protein interactions graphene energy transfer dynamic structural biology super-resolution microscopy

Single-Molecule Dynamic Structural Biology: Breakthrough in Observing DNA-Protein Interactions Using Graphene

Background Introduction

The intricate and precise interactions between DNA and proteins play a crucial role in fundamental biological functions such as DNA replication, transcription, and repair. However, observing these interaction processes in detail has proven challenging, particularly when attempting to capture structural changes at molecular scale (nanometer or even angstrom levels). Traditional structural biology techniques, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and electron microscopy, though offering high resolution, often require sample fixation or manipulation, making it difficult to capture dynamic behaviors of molecular movements under physiologically relevant conditions. Additionally, single-molecule fluorescence resonance energy transfer (smFRET) has provided an essential tool for dynamic structural biology; yet, it is limited to measuring pairwise molecular distances and poses challenges regarding resolution and scalability.

To overcome the limitations of existing methods, the authors of this paper have developed a novel experimental approach called “Graphene Energy Transfer with Vertical Nucleic Acids” (GETVNA). This method enables real-time studies of DNA conformational changes and their interactions with proteins at sub-nanosecond temporal resolution and angstrom-scale spatial resolution. By designing vertically arranged DNA fragments and leveraging their energy transfer properties with graphene, the researchers succeeded in observing dynamic molecular interactions.

Source of the Paper

This study was collaboratively conducted by scientists from institutions including Ludwig-Maximilians-Universität München, University of Illinois at Urbana-Champaign, Rudolf Virchow Center, and the Institute of Physical Chemistry of the Polish Academy of Sciences. The paper was published in the January 2025 issue of Nature Methods, under the title “Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope,” and was made available online on November 8, 2024.

Research Process in Detail

1. Experimental Design and Methodological Innovations

The paper employs a novel graphene energy transfer (GET) technique to study the dynamic processes of DNA-protein interactions. The core of the method lies in the spontaneous vertical alignment of DNA fragments through single-stranded DNA (ssDNA) overhangs, which are anchored to graphene surfaces, and in utilizing the energy transfer relationship between fluorescent probes and graphene to measure DNA conformational changes. The specifics include:

  1. DNA Construction and Vertical Arrangement: Double-stranded DNA (dsDNA) fragments containing ssDNA overhangs are used. Through base stacking interactions, the bottom part of the DNA is firmly adsorbed onto the graphene surface, naturally creating a vertical arrangement.

  2. Fluorescence Lifetime Measurements: Fluorescent dyes (e.g., Atto 542) are attached to the top of DNA fragments. Changes in fluorescence lifetime are measured to calculate the distance between the dye and the graphene (i.e., the “height” of the DNA).

  3. Vertical Positioning Precision: The experiment achieves subnanometer axial localization precision ( Å) by leveraging the energy transfer properties of graphene. Fluorescence lifetime was preferred over intensity measurements for increased robustness against environmental fluctuations.

2. Experimental Steps and Data Collection

  1. Graphene Preparation and Treatment: High-quality single-layer graphene was applied to glass substrates, followed by a series of cleaning and heat treatment steps to ensure surface uniformity.

  2. Validation with Molecular Dynamics Simulations: Molecular dynamics (MD) simulations were conducted to model the adsorption conformation and thermodynamic properties of DNA on graphene. The results confirmed that the DNA maintained its vertical stability, with the bottom base pairs firmly anchored to graphene.

  3. Dynamic DNA Conformation Monitoring:

    • Classical Conformation Analysis: Heights of DNA fragments of different lengths (36 bp, 51 bp, 66 bp) were measured, and the experimental values closely matched theoretical models such as the worm-like chain model.
    • DNA Bending Tests: By introducing base defects, such as adenine tracts or bulges, or via enzyme binding (e.g., Endonuclease IV), DNA bending was induced, and precise bending angles (° accuracy) were extracted from time trajectories.

  4. Protein Diffusion Observation: In studying the diffusion of O6-alkylguanine DNA alkyltransferase (AGT) along DNA, GETVNA achieved single base-pair resolution in real-time tracking.

Major Research Results

Dynamic DNA Conformation Analysis

The authors demonstrated that GETVNA could precisely capture subtle DNA bending and conformational changes. Examples include: - At a location with an adenine tract (A-tract, consisting of 7 adenines), DNA exhibited a bending angle of 33.5°, with measurement accuracy exceeding that of existing FRET methods. - DNA samples with bulges (e.g., 3 unpaired adenines) showed multi-state bending structures, with angles ranging from 23° to 82°. - Molecular dynamics simulations were used to complement experimental results and explain the various conformational states of DNA bending.

Enzyme-Induced Bending and Conformational Changes

  • Upon the addition of Endonuclease IV (E. coli), significant bending of DNA at AP sites was observed (main angle approximately 67°), with dynamic transitions between states captured in real-time.
  • Compared to static structures from crystallography, GETVNA unveiled additional dynamic states and their transitions.

Protein Diffusion Resolution and Dynamics

Using GETVNA, the authors achieved angstrom-scale resolution of AGT diffusion along DNA and quantified single base-pair movements (~3.4 Å step size), offering new insights into molecular motors and their interactions with DNA.

Significance and Value of the Research

Scientific Value

This novel technology provides an important exploratory tool for dynamic structural biology at the molecular scale, bridging existing gaps between dynamic processes and real-time observation: 1. Achieving angstrom-scale measurements and single base-pair tracking under physiologically relevant conditions. 2. Capturing DNA conformational flexibility and dynamic behaviors, offering a dynamic perspective on nucleic acid-protein interactions.

Applications

  • The technique can be broadly applied to studies of DNA repair, transcriptional regulation, and nucleosome assembly.
  • It can be extended to RNA structure and protein-nucleic acid complexes and may hold potential for the development of conductive devices such as field-effect transistors and graphene biosensors.

Study Highlights

  1. Methodological Breakthrough: GETVNA uniquely combines graphene energy transfer with axial localization precision, creating a breakthrough in dynamic DNA measurements.
  2. Diverse Results: Multiple previously unobserved intermediate state dynamics of DNA structures were captured.
  3. Simplified Experiment Design: Only single-label fluorescent probes are needed, avoiding coating modifications or complex reactions, with straightforward interpretation of results.

This study presents a groundbreaking approach for observing dynamic structural biology of DNA and offers a powerful tool for understanding biomolecular interaction processes. Its potential applications span structural biology, molecular biology, and nanotechnology, heralding impactful advances across these fields.