9 research outputs found
Next-Generation Protein Identification: Advancing Single-Molecule Fluorescence Approaches
Proteins are the workhorses of the cell, as such, they form the basis of all living systems. In order to fully understand biological processes, the ability to identify and quantify the proteins in cells is crucial. Identification can be achieved by determining the amino acid sequence of proteins, since this sequence is unique for each protein. However, protein sequencing remains an enormous challenge. The dynamic range at which proteins can occur spans several orders of magnitude, and the need to identify all 20 different amino acids are only a few of the challenges that are currently preventing us from sequencing proteins. However, when realized, single-molecule protein sequencing will create the opportunity for single-cell proteomics and screening for on-site medical diagnostics. It will lead to a revolution in biophysics, biotechnology, and healthcare. Fluorescence techniques belong to one of the most commonly used techniques in biophysics and have brought about a deeper understanding of biological processes at the single-cell or single-molecule level. Furthermore, the field of DNA sequencing has demonstrated that the use of fluorescence approaches has enabled one of the main breakthroughs in DNA sequencing: the development of next-generation sequencers (NGS). These NGS greatly reduced the sequencing costs per human genome. It comes as no surprise that fluorescence approaches are being explored for protein sequencing as well. In this thesis, we pioneer with single-molecule FRET (fluorescence resonance energy transfer) and DNA nanotechnology approaches for the development of a protein identification platform.BN/Chirlmin Joo La
Completing the canvas: advances and challenges for DNA-PAINT super-resolution imaging
Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly smaller structures. At the forefront of these developments is DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), which exploits the stochastic and transient binding of fluorescently labeled DNA probes. In its early stages the implementation of DNA-PAINT was burdened by low-throughput, excessive acquisition time, and difficult integration with live-cell imaging. However, recent advances are addressing these challenges and expanding the range of applications of DNA-PAINT. We review the current state of the art of DNA-PAINT in light of these advances and contemplate what further developments remain indispensable to realize live-cell imaging.Accepted Author ManuscriptBN/Chirlmin Joo La
High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)
Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures. BN/Chirlmin Joo La
FRETboard: semisupervised classification of FRET traces
Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility to adapt to different experimental setups and require local installations. Here, we propose to fit models to optical signals more intuitively by adopting a semisupervised approach, in which the user interactively guides the model to fit a given data set, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios and correctly estimate parameters for up to 11 states. On in vitro data, we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.Accepted Author ManuscriptBN/Chirlmin Joo La
Evaluation of FRET X for single-molecule protein fingerprinting
Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.BN/Chirlmin Joo La
Light-Sensitive Phenacyl Crosslinked Dextran Hydrogels for Controlled Delivery**
Stimuli-responsive soft materials enable controlled release of loaded drug molecules and biomolecules. Controlled release of potent chemotherapeutic or immunotherapeutic agents is crucial to reduce unwanted side effects. In an effort to develop controlled release strategies that can be triggered by using Cerenkov luminescence, we have developed polymer hydrogels that can release bovine serum albumin and immunoglobulin G by using light (254 nm–375 nm) as a trigger. We describe the synthesis and photochemical characterization of two light sensitive phenacyl bis-azide crosslinkers that are used to prepare transparent self-supporting hydrogel patches. One crosslinker was designed to optimize the overlap with the Cerenkov luminescence emission window, bearing an π-extended phenacyl core, resulting in a high quantum yield (14 %) of photocleavage when irradiated with 375 nm light. We used the extended phenacyl crosslinker for the preparation of protein-loaded dextran hydrogel patches, which showed efficient and selective dosed release of bovine serum albumin or immunoglobulin G after irradiation with 375 nm light. Cerenkov-triggered release is as yet inconclusive due to unexpected side-reactivity. Based on the high quantum yield, efficient release and large overlap with the Cerenkov window, we envision application of these photosensitive soft materials in radiation targeted drug release.ChemE/Advanced Soft MatterBN/Chirlmin Joo LabImPhys/OpticsServicedesk TNWRST/Applied Radiation & Isotope
High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT
Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.BN/Chirlmin Joo LabBN/Cees Dekker LabBN/Technici en Analiste
Conditional Copper-Catalyzed Azide–Alkyne Cycloaddition by Catalyst Encapsulation
Supramolecular encapsulation is known to alter chemical properties of guest molecules. We have applied this strategy of molecular encapsulation to temporally control the catalytic activity of a stable copper(I)–carbene catalyst. Encapsulation of the copper(I)–carbene catalyst by the supramolecular host cucurbit[7]uril (CB[7]) resulted in the complete inactivation of a copper-catalyzed alkyne–azide cycloaddition (CuAAC) reaction. The addition of a chemical signal achieved the near instantaneous activation of the catalyst, by releasing the catalyst from the inhibited CB[7] catalyst complex. To broaden the scope of our on-demand CuAAC reaction, we demonstrated the protein labeling of vinculin with the copper(I)–carbene catalyst, to inhibit its activity by encapsulation with CB[7] and to initiate labeling at any moment by adding a specific signal molecule. Ultimately, this strategy allows for temporal control over copper-catalyzed click chemistry, on small molecules as well as protein targets.ChemE/Advanced Soft MatterBN/Chirlmin Joo LabBT/Biocatalysi
The emerging landscape of single-molecule protein sequencing technologies
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.Accepted Author ManuscriptChemE/Advanced Soft MatterBN/Chirlmin Joo LabBN/Cees Dekker La