175 research outputs found
Making connections—strategies for single molecule fluorescence biophysics
Fluorescence spectroscopy and fluorescence microscopy
carried out on the single molecule level are elegant methods to
decipher complex biological systems; it can provide a wealth of
information that frequently is obscured in the averaging of
ensemble measurements. Fluorescence can be used to
localise a molecule, study its binding with interaction partners
and ligands, or to follow conformational changes in large
multicomponent systems. Efficient labelling of proteins and
nucleic acids is very important for any fluorescence method,
and equally the development of novel fluorophores has been
crucial in making biomolecules amenable to single molecule
fluorescence methods. In this paper we review novel coupling
strategies that permit site-specific and efficient labelling of proteins. Furthermore, we will discuss progressive single molecule approaches that allow the detection of individual molecules and biomolecular complexes even directly isolated from cellular extracts at much higher and much lower concentrations than has been possible so far
DNA-Liposome Hybrid Carriers for Triggered Cargo Release
The design of simple and versatile synthetic routes to accomplish triggered-release properties in carriers is of particular interest for drug delivery purposes. In this context, the programmability and adaptability of DNA nanoarchitectures in combination with liposomes have great potential to render biocompatible hybrid carriers for triggered cargo release. We present an approach to form a DNA mesh on large unilamellar liposomes incorporating a stimuli-responsive DNA building block. Upon incubation with a single-stranded DNA trigger sequence, a hairpin closes, and the DNA building block is allowed to self-contract. We demonstrate the actuation of this building block by single-molecule Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching, and fluorescence quenching measurements. By triggering this process, we demonstrate the elevated release of the dye calcein from the DNA-liposome hybrid carriers. Interestingly, the incubation of the doxorubicin-laden active hybrid carrier with HEK293T cells suggests increased cytotoxicity relative to a control carrier without the triggered-release mechanism. In the future, the trigger could be provided by peritumoral nucleic acid sequences and lead to site-selective release of encapsulated chemotherapeutics. © 2022 American Chemical Society. All rights reserved
Single antibody detection in a DNA origami nanoantenna
DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm
Distance dependence of single-molecule energy transfer to graphene measured with DNA origami nanopositioners
Despite the thorough investigation of graphene since 2004, altering its surface chemistry and reproducible functionalization remain challenging. This hinders fabrication of more complex hybrid materials with controlled architectures, and as a consequence the development of sensitive and reliable sensors and biological assays. In this contribution, we introduce DNA origami structures as nanopositioners for placing single dye molecules at controlled distances from graphene. The measurements of fluorescence intensity and lifetime of single emitters carried out for distances ranging from 3 to 58 nm confirmed the d–4 dependence of the excitation energy transfer to graphene. Moreover, we determined the characteristic distance for 50% efficiency of the energy transfer from single dyes to graphene to be 17.7 nm. Using pyrene molecules as a glue to immobilize DNA origami nanostructures of various shape on graphene opens new possibilities to develop graphene-based biophysics and biosensing
DNA origami-based single-molecule force spectroscopy elucidates RNA Polymerase III pre-initiation complex stability
The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important
constituents of all eukaryotic initiation complexes. The reason for the emergence and
strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III
system, however, remained elusive. A poorly studied aspect in this context is the effect of
DNA strain arising from DNA compaction and transcriptional activity on initiation complex
formation. We made use of a DNA origami-based force clamp to follow the assembly of
human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level
under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and
TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal
component that ensures stable anchoring of initiation factors, and thus the polymerase itself,
in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the
high transcriptional output of RNAP II
Plasmon-assisted Förster resonance energy transfer at the single-molecule level in the moderate quenching regime
Metallic nanoparticles were shown to affect Förster energy transfer between fluorophore pairs. However, to date, the net plasmonic effect on FRET is still under dispute, with experiments showing efficiency enhancement and reduction. This controversy is due to the challenges involved in the precise positioning of FRET pairs in the near field of a metallic nanostructure, as well as in the accurate characterization of the plasmonic impact on the FRET mechanism. Here, we use the DNA origami technique to place a FRET pair 10 nm away from the surface of gold nanoparticles with sizes ranging from 5 to 20 nm. In this configuration, the fluorophores experience only moderate plasmonic quenching. We use the acceptor bleaching approach to extract the FRET rate constant and efficiency on immobilized single FRET pairs based solely on the donor lifetime. This technique does not require a posteriori correction factors neither a priori knowledge of the acceptor quantum yield, and importantly, it is performed in a single spectral channel. Our results allow us to conclude that, despite the plasmon-assisted Purcell enhancement experienced by donor and acceptor partners, the gold nanoparticles in our samples have a negligible effect on the FRET rate, which in turns yields a reduction of the transfer efficiency
Strong plasmonic enhancement of single molecule photostability in silver dimer optical antennas
Photobleaching is an effect terminating the photon output of fluorophores, limiting the duration of fluorescence-based experiments. Plasmonic nanoparticles (NPs) can increase the overall fluorophore photostability through an enhancement of the radiative rate. In this work, we use the DNA origami technique to arrange a single fluorophore in the 12-nm gap of a silver NP dimer and study the number of emitted photons at the single molecule level. Our findings yielded a 30× enhancement in the average number of photons emitted before photobleaching. Numerical simulations are employed to rationalize our results. They reveal the effect of silver oxidation on decreasing the radiative rate enhancement.We acknowledge funding by a starting
grant (SiMBA, EU 261162) of the European Research
Council (ERC) and the Deutsche Forschungsgesellschaft
(AC 279/2-1 and TI 329/9-1). IK is grateful for the support
by the Mobility Plus grant 1269/MOB/IV/2015/0 from
the Polish Ministry of Science and Higher Education
(MNiSW). CV thanks a scholarship of the Studienstiftung
des deutschen Volkes. AIF-D and AC-G acknowledge funding
from the Spanish MINECO under Contracts FIS2015-
64951-R and MDM-2014-0377-16-4, respectively. AIF-D
also acknowledges funding from EU Seventh Framework
Programme under Grant Agreement FP7-PEOPLE-
2013-CIG-630996. GA and PT acknowledge funding of the
state ministry for research of lower saxony in the frame of
the “Quantum- and Nanometrology” (QUANOMET) strategic
research area. Quanomet is part of the LUH-TUBS
research allianc
Directing single-molecule emission with dna origami-assembled optical antennas
We demonstrate the capability of DNA self-assembled optical antennas to direct the emission of an individual fluorophore, which is free to rotate. DNA origami is used to fabricate optical antennas composed of two colloidal gold nanoparticles separated by a predefined gap and to place a single Cy5 fluorophore near the gap center. Although the fluorophore is able to rotate, its excitation and far-field emission is mediated by the antenna, with the emission directionality following a dipolar pattern according to the antenna main resonant mode. This work is intended to set out the basis for manipulating the emission pattern of single molecules with self-assembled optical antennas based on colloidal nanoparticles
Addressable Nanoantennas with Cleared Hotspots for Single-Molecule Detection on a Portable Smartphone Microscope
The advent of highly sensitive photodetectors1,2 and the development of photostabilization strategies3 made detecting the fluorescence of a single molecule a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g. in point-of-care diagnostic settings, where costly equipment would be prohibitive.4 Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in the NACHOS emit up to 461-fold brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia "on the road “
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